Mammalian inward rectifier potassium channel cDNA, IRK1, corresponding vectors, and transformed cells

ABSTRACT

This disclosure relates to two separate and distinct inward rectifier K +   channel expression products and the the genes which encode each expression product. The IRK1 gene (SEQ. ID NO: 1) encodes an inward rectifier K +   channel and the GIRK1 gene (SEQ. ID NO: 31) encodes a G protein coupled muscarinic K +   channel. The disclosure relates to the uses of these expression products, particularly in combination with identifying physiological processes mediated by these channels, such as regulation of heartbeat and insulin release and materials modulating or blocking same.

ACKNOWLEDGEMENT

This invention was made with Government support under Grant No.P50MH48200 awarded by the National Institute of Health. The Governmenthas certain rights in this invention. This invention was also made withsupport from the Muscular Dystrophy Association, Inc.

FIELD OF THE INVENTION

The invention relates generally to advances in the field of cellphysiology, specifically, physiologic and recombinant methods useful tocharacterize cell function and physiology. Particularly, the inventionrelates to the molecular identity of two particular and distincttrans-membrane ion channels, the definition of the biophysicalproperties of these channels, the expression products of the genes whichencode these channels and uses of the same. More particularly, theinvention arises in part from the determination that the DNA sequence ofthe IRK1 gene (SEQ. ID NO: 1) encodes an inward rectifier potassiumchannel and the DNA sequence of the GIRK1 gene (SEQ. ID NO: 31) encodesa G protein coupled muscarinic potassium channel.

BACKGROUND OF THE INVENTION

The membranes found at the surface of mammalian cells perform functionsof great importance relating to the integrity and activities of cellsand tissues. Of particular interest is the study of ion channelbiochemistry, physiology, pharmacology and biokinetics. These ionchannels, which include sodium (Na⁺), potassium (K⁺) and calcium (Ca²⁺)channels are present in all mammalian cells and control a variety ofphysiological and pharmacological processes.

Potassium channels are implicated in a broad spectrum of processes inexcitable and non-excitable cells. These physiologic processes includeregulation of heartbeat (Breitwieser, G. E. and Szabo, G. (1985) Nature317:538, Logothetis, D. E. et al. (1987) Nature 325:321, Yatani, A. etal. (1987) Science 235:207, Yanti, A. et al. (1988) Nature 336:680,Brown, A. M. and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52:197 andKurachi, Y. et al. (1992) Progress in Neurobiol. 39:229), dilation ofarteries (Nelson, M. T. et al. (1990) Nature 344:770), release ofinsulin (Rorsman, P. et al. (1991) Nature 349:77), excitability of nervecells (Stanfield, P. R. et al. (1985) Nature 315:498, Williams, J. T. etal. (1988) J. Neurosci. 8:3499) and regulation of renal electrolytetransport (Wang, W. et al. (1992) Annu. Rev. Physiol. 54:81).

Several classes of K⁺ channels have been identified based on theirpharmacological and electrophysiological properties; these includevoltage-gated, ATP-sensitive, muscarinic-activated, S type, SK Ca²⁺-activated, Na⁺ -activated and inward rectifier types of K⁺ channels(For review see Hille, B. Ionic Channels of Excitable Membranes 2d ed.,Sinauer, Sunderland, Mass., 1992).

The best characterized class of K⁺ channels are the voltage-gated K⁺channels. The prototypical member of this class is the protein encodedby the Shaker gene in Drosophila melanogaster (Papazian, D. M. et al.(1987) Science 237:749, Tempel, B. L. et al. (1987) Science 237:770 andBaumann, A. et al. (1987) EMBO J. 6:3419). Mammalian homologues of theDrosophila Shaker and related Shal, Shab and Shaw genes have been cloned(Wei, A. et al. (1990) Science 248:599, Tempel, B. L., Jan, Y. N. andJan, L. Y. (1988) Nature 332:837, Baumann, A. et al. (1988) EMBO J.7:2457, Frech, G. C. et al. (1989) Nature 340:642, Yokoyama, S. et al.(1989) FEBS Lett. 259:37, Cristie, M. J. et al. (1989) Science 244:221,Stuhmer, W. et al. (1989) EMBO J. 8:3235, Swanson, R. et al. (1990)Neuron 4:929 and Luneau, C. J. et al. (1991) Proc. Natl. Acad. Sci. USA88:3932). Voltage-gated K⁺ channels belong to the superfamily ofvoltage-gated and second messenger-gated cation channels (Jan L. Y. andJan, Y. N. (1992) Cell 69:715 and Jan, L. Y. and Jan, Y. N. (1990)Nature 345:672). The proteins in this gene family contain one or fourcopies of an underlying structural motif characterized by sixmembrane-spanning segments (S1-S6), a putative voltage sensor (S4) andan S5-S6 linker (H5 or P region) involved in ion conduction. The vastmajority of cloned K⁺ channels share a structural organization to theabove motif, thereby placing most of the cloned K⁺ channels in the sameK⁺ channel superfamily. Only two cloned K⁺ channels do not share theabove structural organization. These are the mink channel (Takumi, T. etal. (1988) Science 242:1042) and the ROMK1 channel, an ATP-regulated K⁺channel (Ho, K. et al. (1993) Nature 362:31). Attempts to isolate inwardrectifier K⁺ channels using sequences derived from members of thevoltage-gated K⁺ channel gene family as probes have been unsuccessful.This suggests that the structural organization of the inward rectifierchannels differs significantly from that of the voltage-gated K⁺channels.

The molecular features of the proteins which comprise the classes of K⁺channels which are not voltage-gated are, for the most part, unknownalthough pharmacological and physiological characteristics have beenelucidated. Of particular interest are the inward rectifier K⁺ channels.

Inward rectifier K⁺ channels allow primarily K⁺ influx but little K⁺outflux. These K⁺ channels have been found in a variety of cell typesincluding skeletal (Katz, B. (1949) Arch. Sci. Physiol. 2:285 andStanden, N. B. and Stanfield, P. R. (1978) J. Physiol. 280:169) andcardiac (Sakmann, B. and Trube, G. (1984) J. Physiol. 347:641) musclecells, starfish and tunicate oocytes (Hagiwara, S. et al. (1976) J. Gen.Physiol. 67:621 and Okamoto, H. et al. (1976) J. Physiol. 254:607),neurons (Mayer, M. L. & Westbrook, G. L. (1983) J. Physiol. 340:19,Mihara, S. et al. (1987) J. Physiol. 390:335, Inoue, M. et al. (1988) J.Physiol. 407:177 and Williams, J. T. et al. (1988) J. Neurosci. 8:3499),glial cells (Barres, B. A. (1991) Current Opinion in Neurobiol. 1:354),blood cells (McKinney, L. C. and Gallin, E. K. (1988) J. Memb. Biol.103:41 and Lewis, D. L. et al. (1991) FEBS Lett. 290:17) and endothelialcells (Silver M. R. & DeCoursey T. E. (1990) J. Gen. Physiol. 96:109).

The inward rectifier K⁺ channels have significant roles in maintainingthe resting potential and in controlling excitability of a cell. Thephysiological functions of the inward rectifier K⁺ channels stem fromtheir unique rectification property and consist of three parts (Hille,B. Ionic Channels of Excitable membranes, 2d ed., Sinauer, Sunderland,Mass., 1992). First, the absence of outward conductance at highlydepolarized membrane potentials allows a cell that expressespredominantly the inward rectifier to maintain prolonged depolarization.This is important for the generation of prolonged action potentials inheart ventricular cells, and for the prevention of double fertilizationof oocytes (Hagiwara, S. and Jaffe, L. A. (1979) Annu. Rev. Biophys.Bioeng. 8:385). Second, the large inward conductance at membranepotentials below the K⁺ equilibrium potential (E_(K)) prevents excessivehyperpolarization, which may be caused by the electrogenic Na⁺ pump(Hille, B., supra). Third, the slight outward conductance of inwardrectifier K⁺ channels at membrane potentials just above E_(K) helps tokeep the resting membrane potential close to E_(K). Modulation of thisconductance level changes the resting potential and alters theexcitability of the cell. For example, it is well known that theactivation of a particular type of inward rectifier K⁺ channel, themuscarinic K⁺ channel, by acetylcholine causes hyperpolarization of thecardiac pacemaker cells and slows the heartbeat (Noma, A. et al. (1979)Pflugers Arch. 381:255).

The inward rectification properties essential for the physiologicalfunctions of these K⁺ channels have been characterized at themechanistic level. These channels are permeable to an inward flow of K⁺ions at membrane potentials below E_(K), which may be varied by changingthe extracellular K⁺ ion concentration (Hagiwara, S. et al. (1976) J.Gen. Physiol. 67:621). Therefore, the inward rectifier K⁺ channels donot activate over a fixed range of membrane potentials, unlikevoltage-gated K⁺ channels. The inward rectification has been shown to bemainly due to the blockade of outward current by internal Mg²⁺ ; in theabsence of Mg²⁺, inward rectifier K⁺ channels exhibit a linearcurrent-voltage relation (Matsuda, K. et al. (1987) Nature 325:156,Vandenberg, C. A. (1987) Proc. Natl. Acad. Sci. USA 84:2560 and Matsuda,H. (1988) J. Physiol. 397:237).

The extensive interaction of the inward rectifier K⁺ channel pore withpermeant ions and blocking ions has been well documented. These studiesreveal that the inward rectifier K⁺ channel has a long-pore withmultiple binding sites; permeant ions enter the pore in single file andexhibit discernible interactions with other ions that either permeate orblock the pore (Hille, B., supra, Hagiwara, S. et al. 1977) J. Gen.Physiol. 70:269 and Ohmori, H. (1980) J. Memb. Biol. 53:143).Interactions between permeant ions are manifested by the fact that K⁺conductance of the inward rectifier does not increase linearly with K⁺concentration (Sakmann, B. and Trube, G., supra). Extracellular cationssuch as barium (Ba²⁺) and cesium (Cs⁺) block the inward rectifier in amanner that depends both on the voltage and on the time elapsedfollowing channel activation. This suggests that these blocking ionsenter the open channel pore so that they sense part of the voltage dropacross the membrane (Hagiwara, S. et al. (1976) J. Gen. Physiol.67:621). The steepness of this voltage dependence of the block furtherindicates that there are multiple binding sites for Cs⁺ in the channelpore (Hagiwara, S. et al. (1976) J. Gen. Physiol. 67:621 and Hille, B.,supra).

Ion channel function can be regulated by various substances, such ashormones and neurotransmitters, via specific membrane receptors. Twomajor categories of receptor-operated ion channels are known. One classconsists of ion channels which have an intrinsic sensor; the receptorsite and a channel pore are present in the same polypeptide. The otherclass consists of ion channels having a remote sensor; the receptor siteand the ion channel are different membrane proteins. G Protein is ofteninvolved in the remote-sensing ion channel models of transmembranesignalling.

An important receptor involved in the regulation of heart rate, themuscarinic receptor, slows heart rate upon activation by parasympatheticnerve stimulation (Trautwein, W. and Dudel, J. (1958) Pflugers Arch.266:324, Noma, A. et al. (1979) Pflugers Arch. 381:255, Sakmann, B. etal. (1983) Nature 303:250 and Soejima, M. and Noma, A. (1984) PflugersArch. 400:424). The heart rate is slowed by the opening of themuscarinic K⁺ channel. This ion channel has a remote sensor as themuscarinic receptor and K⁺ channel exist as separate protein molecules.

The muscarinic K⁺ channels in the sinoatrial node and atrium, thepacemaker of the heart, are inward rectifying K⁺ channels and are knownto be directly coupled with G proteins (Breitwieser, G. E. and Szabo, G.(1985) Nature 317:538, Logothetis, D. E. et al. (1987) Nature 325:321,Latani, A. et al. (1987) Science 235:207, Latani, A. et al. (1988)Nature 336:680, Brown, A. M. and Birnbaumer, L. (1990) Annu. Rev.Physiol. 52:197 and Kurachi, Y. et al. (1992) Progress in Neurobiol.39:229). G proteins are a class of proteins involved in intracellularsignal transduction. The G protein senses when a ligand has occupied acell surface receptor, binds GTP and activates another protein involvedin the signal transduction pathway such as adenyl cyclase or an ionchannel. In the case of the muscarinic receptor, the activated G proteinopens the muscarinic K⁺ channel causing an outflux of K⁺ from the heartmuscle cell.

While mechanistic studies have elucidated physiological andpharmacological properties of the muscarinic K⁺ channel, no studies havebeen possible at a molecular level. The molecular cloning of themuscarinic K⁺ channel would allow the development of assay systems toidentify compounds which selectively inhibit this ion channel therebyproviding compounds useful for the regulation of heart rate in mammals.

The art needs molecular characterization of inward rectifier K⁺ channelsin order to elucidate the physiological functions and biophysicalproperties of these channels. An understanding of these properties willallow the regulation of the physiological functions performed by theseK⁺ channels, such as regulation of heartbeat and release of insulin.Additionally, the availability of gene sequences encoding these inwardrectifier K⁺ channels would enable assay systems which would allow theidentification of materials capable of selectively blocking thesechannels. Presently compounds which effect K⁺ channels are identifiedusing a cell or a tissue in which multiple types of K⁺ channels arepresent; accordingly it is not possible to determine that a givencompound exerts its effect solely through its interaction with a giventype of K⁺ channel in the present assay. Indeed the K⁺ channelmodulating drugs currently used to treat physiological disordersmediated by a given class of K⁺ channels often have undesirable sideeffects. The art needs a means to identify compounds which have aspecific and selective effect on a single type of K⁺ channel for theimproved treatment of disease.

SUMMARY OF THE INVENTION

The present invention is grounded in the unequivocal finding that theIRK1 gene encodes an inward rectifier K⁺ channel in mouse cells. Thepresent invention is further grounded in the unequivocal finding thatthe GIRK1 gene (SEQ. ID NO: 3) encodes a G protein coupled muscarinic K⁺channel, an inward rectifier K⁺ channel, in rat cells. Further researchrevealed the finding that functional inward rectifier K⁺ channelexpression products of encoding DNA of the IRK1 gene, (SEQ. ID NO: 1) orfunctionally bioactive equivalents, provided the means for developingassays, methods and products for use pharmacologically in animals(including not only the rodent order), and homologously in human beings.Further research also revealed the finding that functional G proteincoupled muscarinic K⁺ channel expression products of encoding DNA of theGIRK1 gene, (SEQ. ID NO: 1) or functionally bioactive equivalents,provided the means for developing assays, methods and products for usepharmacologically in animals (including not only the rodent order), andhomologously in human beings.

Thus, the invention provides an assay for identifying materials having amodulating effect on inward rectifier K⁺ channels, including the Gprotein coupled muscarinic K⁺ channel, in a mammal which comprises thesteps of: providing an expression system that produces a functionalinward rectifier K⁺ channel expression product from DNA sequencesencoding a mammalian inward rectifier K⁺ channel; contacting theexpression system or the product of the expression system or itsequivalent with one or more of a battery of test materials that canpotentially modulate the bioactivity of the expression product or itsequivalent and monitoring the effect of the test materials on theexpression product or its equivalent and selecting a candidate orcandidates from the battery of test materials capable of modulating thebioactivity of the inward rectifier K⁺ channels.

The selecting step of the assay may preferably measure the capacity ofthe test materials to block the bioactivity of the product or itsequivalent. In a preferred embodiment, the IRK1 gene (SEQ. ID NO: 1) maycomprise a mouse inward rectifier K⁺ channel gene. In another preferredembodiment, the GIRK1 gene (SEQ. ID NO: 1) may comprise a rat G proteincoupled muscarinic K⁺ channel gene. Furthermore, the functionallybioactive equivalent may preferably comprise a functional human inwardrectifier or G protein coupled muscarinic K⁺ channel expression product.This functionally active bioequivalent, in a preferred embodiment, maycomprise a functional homologue of a human inward rectifier K⁺ channelexpression product, including a G protein coupled muscarinic K⁺ channelexpression product, which furthermore may comprise a product ofsynthetic derivation. In a preferred embodiment, the expression systemmay comprise a transfectant, the transfectant most preferably comprisinga Xenopus oocyte harboring DNA operatively encoding the product orequivalent.

In accordance with another aspect of the invention, there are providedmethods useful to regulate the release of insulin and to regulate theheartbeat in a mammal. These functions are known to be mediated byinward rectifier K⁺ channels (Nichols, C. G. and Lederer, W. J. (1991)Am. J. Physiol. 261:H1675, Venkatesh, N. et al. (1991) Circ. Res.69:623, and Rorsman, P. et al. (1991) Nature 349:77). These methodscomprise effecting the steps outlined in the above-referenced assay andthen contacting the mammalian cells with the candidates selected in theassay. The contacting step used in these methods is preferablyaccomplished via a composition containing the candidate as an essentialcomponent. Most preferably, the contacting is accomplished viaadministration to a human subject.

In another embodiment of the invention, there is provided, for theabove-disclosed use, DNA (recombinant or cDNA) encoding the IRK1 (SEQ.ID NO: 2) and GIRK1 (SEQ. ID NO: 4) gene products or functionallybioactive equivalents thereof, and vectors and transfectants operativelyharboring same. In a preferred embodiment, the DNA encodes the IRK1 geneproduct (SEQ. ID NO: 2). In another preferred embodiment, the DNAencodes the GIRK1 gene product (SEQ. ID NO: 4). In another preferredembodiment, there is provided a transfected cell comprising DNA encodingthe IRK1 gene product (SEQ. ID NO: 1) or a functionally bioactiveequivalent thereof. In yet another preferred embodiment, there isprovided a transfected cell comprising DNA encoding the GIRK1 geneproduct (SEQ. ID NO: 3) or a functionally bioactive equivalent thereof.

In a particularly preferred embodiment, the transfected cell harboringeither of the inward rectifier K⁺ channel genes is a Xenopus oocyte. Afurther preferred embodiment provides vectors comprising the DNAencoding either the IRK1 or GIRK1 (SEQ. ID NO: 4) gene products (SEQ. IDNO: 2) or functionally bioactive equivalents thereof.

The invention is further directed to isolates of DNA encoding the IRK1(SEQ. ID NO: 2) and GIRK1 (SEQ. ID NO: 4) gene products or functionallybioactive equivalents. It is further directed to expression vectorsharboring such DNA comprising expression control elements operative inthe recombinant host selected for the expression of such DNA andpreferably comprising appropriate initiation (i.e., promoter andenhancer elements), termination, replication, and other sequences thatfunctionally assist the integration of the expression vector into arecombinant host by transfection, optionally coupled with actualintegration into the host's genome.

In respect of the recombinant DNA aspects of the invention, thetechnology is applicable directly in all of its aspects, for example:DNA isolate production; including DNA isolates capable of hybridizing toIRK1 (SEQ. ID NO: 1) or GIRK1 (SEQ. ID NO: 3) gene sequences under lowstringency conditions; devising expression vectors for them; andproducing transfected hosts.

Further, the invention is directed to the foregoing aspects and all oftheir associated embodiments as will be represented as equivalentswithin the skill of those in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a depicts the inward rectifying currents induced in Xenopusoocytes by injection of total poly A⁺ RNA from J774 cells, sizefractionated RNA and water.

FIG. 1b depicts the current-voltage (I-V) curve of the data shown inFIG. 1a.

FIG. 1c depicts the inward rectifying currents induced in Xenopusoocytes by injection of RNA transcribed from the IRK1 cDNA clone.

FIG. 1d depicts the I-V plot of the data shown in FIG. 1c.

FIG. 2 depicts the inward rectifying currents induced in Xenopus oocytesby the IRK1 encoded K⁺ channel and the response to the addition ofexternal Na⁺, Ba²⁺ and Cs⁺.

FIGS. 2a, 2c, 2e and 2g depict traces elicited by steps to +50, +20,-10, -40, -70, -100, -130 and -160 mV in the presence variousconcentrations of the following ions: K⁺(2a), Na⁺ (2c), Ba²⁺ (2e) andCs⁺ (2g).

FIGS. 2b, 2d, 2f and 2h depict I-V plots of the data shown in FIGS. 2a,2c, 2e and 2g, respectively.

FIG. 3a depicts single channel activities of the IRK1 K⁺ channel.Current recordings are shown at the voltages indicted.

FIG. 3b depicts I-V plot of the data shown in FIG. 3a.

FIGS. 4a(1)-(3) shows the nucleotide and deduced amino acid sequence ofIRK1 (SEQ. ID NO: 2).

FIG. 4b shows the alignment of the amino acid sequences of IRK1 (SEQ. IDNO: 2) and the ATP-regulated K⁺ channel (ROMK1) (SEQ. ID NO: 5).

FIGS. 4c-4e depict the alignment of the amino sequences of IRK1 (SEQ. IDNO: 2) and other K⁺ channel sequences in the H5 (4c), (SEQ. ID NO: 6-11)S5 (4d) (SEQ. ID NO: 12-17) and S6 (4e) (SEQ. ID NO: 18-23) regions.

FIG. 5 depicts the distribution of IRK1 mRNA in mouse tissues (upperpanel) and the presence of α-tublin in the same samples as a control forthe integrity of the RNA (lower panel).

FIG. 6 depicts the proposed membrane topology of the IRK1 encoded inwardrectifier K⁺ channel (right) and proposed membrane topology of thevoltage-gated K⁺ channel (left).

FIG. 7a(1)-(3) depicts the nucleotide and deduced amino acid sequence ofGIRK1 (SEQ. ID NO: 3 and 4).

FIG. 7b depicts the alignment of the amino acid sequences of GIRK1,(SEQ. ID NO: 4) IRK1 (SEQ. ID NO: 2) and ROMK1 (SEQ. ID NO: 5).

FIG. 8a depicts current traces from oocytes injected with m2 muscarinicreceptor and GIRK1 cDNA before and after application of carbachol. Thecarbachol-induced currents depicted are the difference between theformer two sets of traces.

FIG. 8b depicts a I-V plot of the carbachol-induced traces shown in FIG.8a.

FIG. 8c depicts current traces from oocytes injected with both GIRK1 andGα_(i2), β₁, γ₂ cDNA, or with either GIRK1 or Gα_(i2), β₁, γ₂ cDNAalone.

FIG. 8d depicts a I-V plot of the data shown in FIG. 8c.

FIG. 8e depicts current traces recorded in 45 mM or 20 mM K⁺ solutionfrom the same oocytes injected with both GIRK1 and Gα_(i2), β₁, γ₂ cDNAas in FIG. 8c.

FIG. 8f depicts a I-V plot of the inwardly rectifying current fromoocytes injected with GIRK1 and Gα_(i2), β₁, γ₂ cDNA in various externalK⁺ concentrations.

FIG. 8g depicts a logarithmic plot of extracellular K⁺ concentrationversus activation potential.

FIG. 9a depicts single channel recordings from oocytes injected withGIRK1 and Gα_(i2), β₁, γ₂ cDNA; one minute segments of continuousrecordings are shown from a membrane in the cell-attached configuration(left panel), inside-out configuration in the absence of GTP (middlepanel) and inside-out in the presence of GTP.sub.γ S (right panel).Expanded traces from the segments indicated by the triangles are shownbelow.

FIG. 9b depicts the open probability measured from the traces shown inFIG. 9a.

FIG. 9c depicts single channel activity in the presence of cytoplasmicMg²⁺ in oocytes injected with GIRK1 and Gα_(i2), β₁, γ₂ cDNA.

FIG. 9d depicts single channel activity of oocytes injected with GIRK1and Gα_(i2), β₁, γ₂ cDNA in cytoplasmic Mg²⁺ -free cytoplasmic solution.

FIG. 9e depicts I-V plots of single channel current from the data shownin FIG. 9c ( ) and 9d (◯).

FIG. 10a shows the distribution of GIRK1 mRNA in rat tissue.

FIG. 10b shows the distribution of GIRK1 mRNA in guinea pig tissue.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

By the term "material" herein is meant any entity that is not ordinarilypresent or functional with respect to inward rectifier K⁺ channels,including G protein coupled muscarinic K⁺ channels, and that effectssame. Thus, the term has a functional definition and includes known, andparticularly, unknown entities that are identified and shown herein tohave a modulating effect on inward rectifier K⁺ channel expression,including G protein coupled muscarinic K⁺ channel expression.

By the term "modulating effect" or grammatical equivalents, herein ismeant both active and passive impact on inward rectifier K⁺ channels,including G protein coupled muscarinic K⁺ channels. These include, butshall not be construed as limited to, blocking the channel or thefunction of the channel protein(s), reducing the number of ion channelsper cell and use of secondary cell(s) or channel(s) to impact on aprimary abnormal cell.

By the term "measuring" in respect of effect of materials on inwardrectifier K⁺ channels, including G protein coupled muscarinic K⁺channels herein is meant any method known or devised for measuring theimpact of a material on said channels/cells. These include, but shallnot be construed as limited to, measuring current, measuring membranepotential, measuring K⁺ flux, such as with radioactive tracers,measuring K⁺ concentration and measurements of indirect consequences toother receptors, second messengers and/or channels.

By the term "functional" in respect of an inward rectifier K⁺ channelexpression product, including a G protein coupled muscarinic K⁺ channelexpression product, herein is meant that product works for its intendedpurpose, to wit, that it is bioreactive equivalently as is the directproduct of either the IRK1 or GIRK1 gene as such.

The term "functionally bioactive equivalent" or "bioreactiveequivalently" or grammatical equivalents thereof refers to mammalianproteins which perform the functions of the expression product encodedby either the IRK1 or GIRK1 gene. These bioactive mammalian proteins orhomologues are capable of producing an expression product whichfunctions as an inward rectifier K⁺ channel of the type encoded byeither the IRK1 or GIRK1 gene.

In a preferred embodiment, the inward rectifier K⁺ channel expressionproduct comprises an amino acid sequence encoded by a nucleotidesequence able to hybridize under low stringency conditions to thecomplement of a nucleotide sequence encoding the protein having theamino acid sequence shown in either FIG. 4a or FIG. 7a.

The amino acid sequence encoded by the cross-hybridizable nucleotidesequence is preferably greater than about 40% homologous, morepreferably greater than about 60% homologous, still more preferablygreater than 70% homologous, even more preferably greater than about80%, and most preferably at least about 90% homologous with the aminoacid sequence shown in either FIG. 4a or FIG. 7a.

"Homologous" is defined as the percentage of residues in the candidateamino acid sequence that are identical with the residues in the aminoacid sequence shown in FIG. 4a or FIG. 7a after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percenthomology.

The terms "nucleic acid molecule encoding", "DNA sequence encoding", and"DNA encoding" refer to the order or sequence of deoxyribonucleotidesalong a strand of deoxyribonucleic acid. The order of thesedeoxyribonucleotides determines the order of amino acids along thepolypeptide (protein) chain. The DNA sequence thus codes for the aminoacid sequence.

The term "isolated" when used in relation to a nucleic acid, as in "aDNA isolate" that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature.However, isolated nucleic acid encoding a mammalian inward rectifier K⁺channel includes such nucleic acid in cells ordinarily expressing aninward rectifier K⁺ channel where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different DNA sequence than that found in nature.

"Low stringency conditions" are overnight incubation at 37° C. in asolution comprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 1× SSC at about 50° C.

By the term "expression system" herein is meant matter capable ofproducing a functional inward rectifier K⁺ channel expression product,including a G protein coupled muscarinic K⁺ channel expression product.In preferred embodiments, such systems are micro-organisms, Xenopusoocytes or cell cultures harboring operatively DNA encoding suchfunctional inward rectifier K⁺ channel expression products, including Gprotein coupled muscarinic K⁺ channel expression products. "Operative,"or grammatical equivalents, means that the respective DNA sequences areoperational, that is, work for their intended purposes. Thus, the DNA ispreferably contained within expression vectors that are used totransfect recombinantly suitable host cells. The vectors and methodsdisclosed herein are suitable for use in host cells over a wide range ofprokaryotic and eukaryotic organisms. "Transfectants" refers to cellsand viruses which have been transfected or transformed with vectorsconstructed using recombinant DNA techniques, including expressionsystems including but not limited to, Xenopus and vaccina.

In general, prokaryotes are preferred for cloning of DNA sequences inconstructing the vectors useful in the invention. For example, E. coliDH10B (Gibco BRL) is particularly useful. Other microbial strains whichmay be used include E. coli strains such as E. coli K12 strain 294 (ATCCNo. 31446), E. coli B, and E. coli X1776 (ATCC No. 31537). Theseexamples are, of course, intended to be illustrative rather thanlimiting.

Prokaryotes may also be used for expression. The aforementioned strains,as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 27235),bacilli such as Bacillus subtilus, and other enterobacteriaceae such asSalmonella typhimurium or Serratia marcestens, and various pseudomonasspecies may be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transfected cells. For example, E. coli istypically transformed using pBR322 or derivatives thereof. pBR322 is aplasmid derived from an E. coli species [Bolivar, et al., Gene 2, 95(1977)]. pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides easy means for identifying transfectedcells. The pBR322 plasmid, or other microbial plasmid must also contain,or be modified to contain, promoters which can be used by the microbialorganism for expression of its own proteins. Those promoters mostcommonly used in recombinant DNA construction include the β-lactamase(penicillinase) and lactose promoter systems [Chang, et al., Nature 275,617 (1978), Itakura, et al., Science 198, 1056 (1977)], Goeddel, et al.,Nature 281, 544 (1970)] and a tryptophan (trp) promoter system [Goeddel,et al., Nucleic Acids Res. 8 4057 (1980); EPO Appl Publ No. 0036776].While these are the most commonly used, other microbial promoters havebeen discovered and utilized, and details concerning their nucleotidesequence have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors [Siebenlist, et al., Cell 20, 269(1980)].

Additionally, phage vectors may be utilized in place of plasmid vectors.Examples of suitable phage vectors are provided in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory, New York, 1989.

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast iscommonly used among eukaryotic microorganisms, although a number ofother strains are commonly available, such as Pichia strains, forexample.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase [Hitzeman, et al., J. Biol. Chem. 255, 12073(1980)] or other glycolytic enzymes [Hess, et al., J. Adv. Enzyme Reg.7, 149 (1968) and Holland, et al., Biochemistry 17, 4000 (1978)], suchas enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. In constructingsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector 3' of thesequences desired to be expressed to provide polyadenylation of the mRNAand termination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionsfor the methanol-regulated alcohol oxidase I (AOX1) gene of Pichiapastoris (see EPA Publn. No. 183071), alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization (Holland, ibid.). Any plasmid vector containing yeastcompatible promoter, origin of replication and termination sequences issuitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be workable, whether from vertebrate orinvertebrate culture. Examples of useful invertebrate cell lines includethe Sf9 cell line (ATCC CRL 1711). However interest has been greatest invertebrate cells, and propagation of vertebrate cells in culture (tissueculture) has become a common procedure in recent years [Tissue Culture,Academic Press, Kruse and Patterson, editors (1973)]. Examples of suchuseful host cell lines are VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, and 293, W138, BHK, COS-7 and MDCK cell lines. Onesuch useful cell line is a CHO line, CHO-K1 ATCC No. CCL 61. Expressionvectors for such cells ordinarily include (if necessary) an origin ofreplication, a promoter located in front of the gene to be expressed,along with any necessary ribosome binding sites, RNA splice sites,polyadenylation site, and transcriptional terminator sequences.

For invertebrate cells, the control functions on the expression vectorsare often provided by viral material derived from Baculovirus. Formammalian cells, the control functions on the expression vectors arealso often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, Rous sarcoma virus,cytomegalovirus, and most frequently Simian Virus 40 (SV40). The earlyand late promoters of SV40 virus are particularly useful because bothare obtained easily from the virus as a fragment which also contains theSV40 viral origin of replication [Fiers, et al., Nature 273, 113(1978)]. Smaller or larger SV40 fragments may also be used, providedthere is included the approximately 250 bp sequence extending from theHind III site toward the Bgl I site located in the viral origin ofreplication. Further it is also possible, and often desirable, toutilize promoter or control sequences ordinarily associated with thedesired gene sequence, provided such sequences are compatible with hostcell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV, etc.) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosomes, the latter is oftensufficient.

If cells without formidable cell membrane barriers are used as hostcells, transfection is carried out by the calcium phosphateprecipitation method as described by Graham and Van der Eb, [Virology52, 456 (1973)]. However, other methods for introducing DNA into cellssuch as by DEAE-dextran mediated transfection, microinjection,electroporation, retroviral infection, lipofection, biolistics or byprotoplast fusion may also be used.

If prokaryotic cells or cells which contain substantial cell wallconstructions are used, the preferred method of transfection is calciumtreatment using calcium chloride as described by Cohen, F. N., et al.,[Proc. Natl. Acad. Sci. (USA) 69, 2110 (1972)] or alternatively,electroporation

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and religated in the formdesired to form the plasmids required.

Cleavage is performed by treating with restriction enzyme (or enzymes)in a suitable buffer. In general, about 1 μl plasmid or DNA fragment isused with about 1 unit of enzyme in about 10-20 μl of buffer solution.(Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer.) Incubation times of about 1hour at 37° C. are workable. After incubations, protein is removed byextraction with phenol and chloroform, and the nucleic acid is recoveredfrom the aqueous fraction by precipitation with ethanol.

If blunt ends are required, the preparation is treated for 15 minutes at15° C. with 10 units of Polymerase I (Klenow), phenol-chloroformextracted, and ethanol precipitated.

Size separation of the cleaved fragments is performed using, forexample, 6 percent polyacrylamide gel described by Goeddel, D., et al.,Nucleic Acids Res. 8, 4057 (1980).

For ligation, approximately equimolar amounts of the desired components,suitable ends tailored to provide correct matching are treated withabout 10 units T4 DNA ligase per 0.5 μg DNA. (When cleaved vectors areused as components, it may be useful to prevent religation of thecleaved vector by pretreatment with bacterial alkaline or calfintestinal phosphatase.)

For analysis to confirm correct sequences in plasmids constructed, theligation mixture may be used to transform E. coli DH10B strain (GibcoBRL) or K12 strain 294 (ATCC No. 31446), and successful transformantsselected by ampicillin, or tetracycline resistance where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionand/or sequenced by the method of Messing, et al., Nucleic Acids Res. 9,309 (1981) or by the method of Maxam, et al., Methods of Enzymology 65,499 (1980).

In addition to the above discussion and the various references toexisting literature teachings, reference is made to standard textbooksof molecular biology that contain definitions and methods and means forcarrying out basic techniques encompassed by the present invention. See,for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual,2d ed., Cold Spring Harbor Laboratory, New York, 1989 and the variousreferences cited therein. All of the herein-cited publications are bythis reference hereby expressly incorporated herein.

The present invention is thus directed to the identification,management, diagnosis and/or control of a disease state including:

(1) selectively screening for preferably selective modulators and/orblocking materials of inward rectifier K⁺ channels, including G proteincoupled muscarinic K⁺ channels, for use as a diagnostic, and/or

(2) blocking, retarding, modulating or eliminating selectively inwardrectifier K⁺ channels, including G protein coupled muscarinic K⁺channels for use as a therapeutic.

B. Preferred Embodiment

The foregoing description and following experimental details set forththe methodology employed initially by the present researchers inidentifying, isolating, characterizing and determining the significanceof the IRK1 (SEQ. ID NO: 2) and GIRK1 (SEQ. ID NO: 4) gene products inrespect of physiological functions mediated via these inward rectifierK⁺ channels. The art-skilled will recognize that by supplying suchinformation for the IRK1 (SEQ. ID NO: 1) and GIRK1 (SEQ. ID NO: 3)genes, as detailed herein, it is not necessary, or perhaps evenscientifically advisable, to repeat those details in their endeavors toreproduce this work. Instead, they may choose to employ alternative,reliable and known methods. Thus, they may identify related polypeptidesvia immuno cross-reactivity to antibodies raised to, for example, itsreactive determinant(s). They may synthesize the underlying DNA sequencefor deployment within similar or other suitable, operative expressionvectors and culture systems. They may use the sequences herein to createprobes, preferably from regions at both the N-terminus and C-terminus,to screen genomic libraries in isolating total encoding DNA fordeployment as described above. They may use the sequence informationherein in cross-hybridization procedures to isolate, characterize anddeploy, as described above, DNA encoding related gene products of otherspecies, or DNA encoding related (e.g., gene family) gene products ofthe same or other species, or to devise DNA for such characterization,use and deployment encoding functionally equivalent gene products of allof the above differing in one or more amino acids from the IRK1 (SEQ. IDNO: 2) or GIRK1 (SEQ. ID NO: 4) gene products or in glycosylationpatterns or in bounded conformational structure.

Alternatively, DNA encoding related gene products of other species, orDNA encoding related (e.g., gene family) gene products of the same orother species may be isolated using the technique of the polymerasechain reaction (PCR). A pair of primers corresponding to two separatestretches of 7 or more highly conserved amino acids from the IRK1 (SEQ.ID NO: 1) or GIRK1 (SEQ. ID NO: 3) gene are synthesized and used in aPCR to isolate gene sequences corresponding to the IRK1 (SEQ. ID NO: 1)or GIRK1 (SEQ. ID NO: 3) genes from other species or to isolate genesequences corresponding to other members of the inward rectifier genefamily.

Thus, in addition to supplying details actually employed, the presentdisclosure serves to enable reproduction of the IRK1 (SEQ. ID NO: 2) andGIRK1 (SEQ. ID NO: 4) gene products disclosed and functionally bioactiveequivalents, using means within the skill of the art having benefit ofthe present disclosure. All of such means are included within theenablement and scope of the invention.

EXAMPLES 1. Cloning of an Inward Rectifier K⁺ Channel

To identify a source for messenger RNA suitable for expression cloningof the inward rectifier, poly A+ RNA was isolated from various tissues(rat brain and skeletal muscle, rat and guinea pig heart, and culturedbovine aortic endothelial cells), and from various cell lines [C2C12 andL6 (myoblast), GH3 (pituitary), GT1-1 (neuroendocrine) and J774(macrophage)]. Xenopus oocytes were injected with about 50 ng of poly A⁺RNA from various sources and membrane currents were recorded under twoelectrode voltage clamp in a 90 mM K⁺ solution, which optimizes thedetection of inward rectifier K⁺ currents. As shown in FIGS. 1a and 1b,poly A⁺ RNA from rat brain, GT1-1 cells and J774 cells induceddetectable expression of an inward rectifier K⁺ current.

The J774 mouse macrophage cells were chosen for the subsequent studies,because the expressed current was large and highly susceptible to blockby external Ba²⁺. The sensitivity of the expressed current to the blockby 100 μM external Ba²⁺ enables it to be distinguished from the variableendogenous inward rectifying currents of the Xenopus oocytes (FIGS. 1aand 1b), which were unaffected by 100 μM external Ba²⁺.

After size fractionation, poly A⁺ RNA of 4.5-5.5 kb induced the largestcurrent (FIGS. 1a and 1b), and was used for the construction of anunidirectional cDNA library.

Poly A+ RNA was isolated from J774 mouse macrophage cells using a FastTrack RNA isolation kit (Invitrogen) and fractionated as described(Meyuhas, O. and Perry, R. P. (1979) Cell 16:139). Briefly, 140 μg polyA⁺ RNA was loaded on a 5-20% sucrose gradient and centrifuged at 21,000rpm for 15.5 hours at 22° C. Thirty fractions were collected andanalyzed electrophysiologically. The size range of RNA in each fractionwas determined by electrophoresis on a formaldehyde agarose gel; poly A⁺RNA was detected with an oligo(dT) probe on the Nytran (S&S) RNA blot.

A cDNA library was constructed essentially as described (Aruffo, A. andSeed, B. (1987) Proc. Natl. Acad. Sci. USA 84:8573). Briefly, the4.5-5.5 Kb enriched RNA fraction was reverse transcribed using MMVSuperscript (BRL) at 42° C. by priming with Notl-oligo(dT) primeradaptors. After ligating a BstX1 adaptor and digestion with Not1, cDNAwas size fractionated on a K⁺ acetate gradient. cDNAs larger than 3 kbwere ligated to a BstX1-Not1 digested pcDNA1/Amp plasmid (Invitrogen).Recombinant plasmids were electroporated into DH10B bacteria (BRL). Theinitial library was composed of 120,000 independent recombinants. Thisprimary library was further size selected by digesting plasmids withNot1, selecting DNA fragments larger than 9 kb (about one quarter of thetotal library), then religating and transforming bacteria. Sixteen poolsof 5,000 recombinants were screened. RNA was transcribed from Not1digested DNA using methylated cap analogue and T7 polymerase asdescribed (Baldwin, T. J. et al. (1991) Neuron 7:471).except that thereaction products were not treated with DNase. Subdivision of a positivepool was repeated until a single clone was obtained.

A single clone (IRK1) isolated from this library, carrying a cDNA of 5.5Kb, was sufficient to give rise to inward rectifier K⁺ current inoocytes (FIGS. 1c and 1d). This K⁺ current resembles the current inducedby total poly A⁺ RNA from J774 cells in its rapid activation belowE_(K).

The electrophysiological studies shown in FIG. 1 were carried out asfollows. Oocytes were injected with 50 nL RNA (1 μg/μl), and treatedwith collagenase for (2 mg/ml) for 2 h at room temperature.Electrophysiological recordings were carried out 48-96 h later at 22°±2°C. by two-electrode voltage clamp (Baldwin, T. J. et al., supra). Dataacquisition and analysis were done on an 80386-based microcomputer usingpclamp program and TL-1A/D converter (Axon Instruments). Microelectrodeswere filled with 3M KCl; the resistance was 0.7-1.4 Mohm. Bath solutioncontained 90 mM KCl, 3 mM MgCl₂, 5 mM HEPES (pH 7.4) (the 90 mM K⁺solution) with 300 μM niflumic acid to block Cl⁻ channels.

The inward rectifying currents induced in oocytes injected with poly A⁺RNA from the J774 cell line (FIG. 1a), water (FIG. 1a),size-fractionated poly A⁺ RNA (FIG. 1a) and transcribed RNA from theIRK1 cDNA clone (SEQ. ID NO: 1) (FIG. 1c) are shown. Currents wererecorded under two-electrode voltage clamp in 90 mM K⁺ solution frominjected oocytes. The holding potential was -30 mV (FIG. 1a), or 0 mV(FIG. 1c). Steps to +50, +20, -10, -40, -70 and -100 mV are shown. Scalebars indicate 500 ms and 1 pA. FIGS. 1b and 1d show I-V curve of thedata shown in FIGS. 1a and 1c, respectively; Current amplitudes justafter the capacitive transient (10 ms from the beginning of the voltagestep) were plotted. Symbols in FIG. 1b represent total poly A⁺ RNA (),4.5-5.5 kb poly A⁺ RNA (); water (); and in FIG. 1d, IRK1 RNA (); water(∇).

2. Electrophysiology of the IRK1 Current

The channel encoded by the IRK1 cDNA clone (SEQ. ID NO: 1) is anauthentic inward rectifier K⁺ channel, because (1) the expressed currentalways activates at membrane potentials below E_(K), (2) the slopeconductance does not increase linearly with external K⁺ concentration;and (3) the current is blocked by external Na⁺, Ba²⁺ and Cs⁺. The singlechannel conductance (21 pS) and inward rectification of the IRK1 singlechannel current are also characteristic of inward rectifier K⁺ channels.

The salient feature of an inward rectifier is its ability to pass largeinward K⁺ current below E_(K) but only minimal outward K⁺ current(Hagiwara, S. et al. (1976) J. Gen. Physiol. 67:621). To investigatethis feature, we varied E_(K) by changing the K⁺ concentration of theexternal solution and measured the activation potential of the IRK1current, which showed strong inward rectification (FIGS. 2a and 2b). Theactivation potential is defined as the potential at which the slopeconductance changes noticeably; the slope conductance is small atpotentials more depolarized than the activation potential and begins toincrease at the activation potential, reaching a high level at morehyperpolarized potentials. The activation potentials measured in threeexperiments were 0±2 mV (90 mM K⁺), -17±2 mV (45 mM K⁺), -4.1±2 mV (20mM K⁺), -56±2 mV (10 mM K⁺) and -75±3mV (4 mM K⁺), which are in goodagreement with E_(K) in these solutions as predicted by the Nernstequation. These results show that the IRK1 channels are K⁺ channelswhich activate at membrane potentials below E_(K) and pass inward K⁺currents.

Another characteristic feature of inward rectifiers is the non-lineardependence of conductance on K⁺ concentration: the slope conductancevaries with the square root of the external K⁺ concentration (Sakmann,B. and Trube, G. (1984) J. Gen. Physiol. 347:641). Indeed, thedouble-logarithmic plot of the slope conductance versus extracellular K⁺concentration (4-20 mM) was fitted with a straight line of slope0.47±0.03 (n=3). Thus, the IRK1 current mimics the inward rectifiercurrents in its deviation from the independence principle, therebyrevealing interactions between K⁺ ions as they go through the channel.

A third characteristic feature of inward rectifiers is theirsusceptibility to the block of the channel pore by extracellular cations(Standen, N. B. and Stanfield, P. R. (1978) J. Physiol. 280:169,Hagiwara, s. et al. (1976) J. Gen. Physiol. 67:621 and Sakmann, B. andTrube, G. (1984) J. Gen. Physiol. 70:269). Such a block was indicated bya prominent inactivation of the IRK1 current in solutions containing 20,10 (FIG. 2a) or 4 mM K⁺ and increased Na⁺ concentrations. Indeed, inexternal solutions of varying Na⁺ concentrations (N-methylglucamine wasused to maintain the ionic strength as the Na⁺ concentration was varied,and the K⁺ concentration was fixed at 4 mM), the inactivation increasedwith the external Na⁺ concentration and was both voltage- andtime-dependent (FIGS. 2c and 2d). In addition to this Na⁺ block, theIRK1 channel was blocked in a voltage- and time-dependent manner byexternal Ba²⁺ (FIGS. 2e and 2f) or Cs⁺ (FIGS. 2g and 2h), suggestingthat all three ions act as open channel pore blockers. The steep voltagedependence of Cs⁺ block was quantitated by first plotting the ratios ofsteady-state current levels in the presence and absence of Cs⁺ as afunction of external Cs⁺ concentration (to obtain K_(i) for the Cs⁺block at each membrane potential), and then fitting the doublelogarithmic plot of K_(i) versus membrane potential with a straightline. A tenfold change in K_(i) corresponded to a change in membranepotential of 38.1 mV. This implies that the fractional distance of theCs⁺ binding site in the membrane electric field is 1.53, if we assumethat only one Cs⁺ enters the pore and blocks K⁺ permeation. Thisapparent anomaly indicates that several Cs⁺ ions reside in a singlechannel pore, as shown previously for inward rectifiers (Hagiwara, S. etal., supra, Hille, B., supra).

The data showing that the IRK1 K⁺ current exhibits characteristic inwardrectification properties and open channel block by external Na⁺, Ba²⁺and Cs⁺ (FIG. 2) was generated as follows. Current recordings undervoltage clamp are shown in FIGS. 2a, 2c, 2e and 2g. Holding potentialwas 0 mV. Traces elicited by steps to +50, +20, -10, -40, -70, -100,-130 and -160 mV are shown. An additional trace elicited byhyperpolarization to -190 mV is shown in FIGS. 2a and 2c. Scale barsindicate 500 ms and 2 μA (FIGS. 2a, 2c, 2e), 100 ms and 2 μA (FIG. 2g).Voltage- and time-dependent block of the IRK1 current is evident in 40and 86 mM Na⁺ (FIG. 2c), 30 μM Ba²⁺ (FIG. 2e) and 30 μM Cs⁺ (FIG. 2g).

FIGS. 2b, 2d, 2f and 2h depict the I-V relations of the data shown inFIGS. 2a, 2c, 2e, 2g, respectively. Current amplitudes just after thecapacitive transient (10 ms from the start of voltage pulses) areplotted in FIG. 2b, and current amplitudes at the end of the 1,600-msvoltage pulses are plotted in FIGS. 2d, 2f and 2h. For the experimentsshown in FIGS. 2a and 2b, current recordings were performed in solutionsof 90 mM (◯), 45 mM () , 20 mM (∇), 10 mM () and 4 mM K⁺ (□) (data notshown in 2a). K⁺ was substituted with Na⁺. E_(K) values in thesesolutions, as predicted by the Nernst equation, were 0 mV (90 mM K⁺),-17.4 mV (45 mM K⁺), -37.9 mV (20 mM K⁺), -55.3 mV (10 mM K⁺) and -78.4mV (4 mM K⁺). (The intracellular K⁺ concentration of oocytes was assumedto be 90 mM, according to previous description in Dascal, N. (1987)Crit. Rev. Biochem. 22:317). IRK1 current in 90 mM K⁺ solution showedslight inactivation shortly after channel activation (within 200 ms),which could be due to block by certain extracellular ions such as Mg²⁺(Biermans, G. et al. (1987) Pflugers Arch. 410:604).

In FIGS. 2c and 2d current recordings were performed in solutions of 0mM (◯), 10 mM (), 20 mM (∇) (data not shown in 2c), 40 mM () and 86 mMNa⁺ (□) solution. All solutions contained 4 mM K⁺, 3 mM Mg²⁺, 5 mM HEPES(pH 7.4). Na⁺ was replaced with N-methylglucamine. The sum of Na⁺ andN-methylglucamine was 86 mM. In FIGS. 2e and 2f current recordings wereperformed in solutions of 0 μM (◯), 3μM (), 30 μM (∇), 300 μM () and 3mM Ba²⁺ (□) (data not shown in 2e). BaCl₂ was added to 90 mM K⁺ solutionwithout correcting the ionic strength. For FIGS. 2g and 2h currentrecordings were performed in solutions of 0 μM (◯), 3 μM (), 30 μM (∇),300 μM () and 3 mM Cs⁺ (□) (data not shown in 2g). CsCl was added to 90mM K⁺ solution without correcting the ionic strength.

By demonstrating K⁺ selectivity, inward rectification and interactionsbetween permeant ions and blocking ions, the results show that the IRK1channel is an inward rectifier K⁺ channel. Apart from the inwardrectifier K⁺ channels, cardiac muscarinic K⁺ channels and ATP-sensitiveK⁺ channels also show moderate inward rectification (Matsuda, H. (1991)J. Physiol. 53:289, Horie, M. and Irisawa, H. (1989) J. Physiol. 408:313and Horie, M. et al. (1987) J. Gen. Physiol. 387:251). The outward K⁺conductances of these two types of channels are much larger than that ofthe IRK1 channel. In addition, the single channel conductance of IRK1channels in cell attached patches was 21±2 pS (n=4) (140 mM external K⁺/˜90 mM intracellular K⁺, at 21°±2° C.) (FIGS. 3a and 3b), which issimilar to that of inward rectifiers (20-30 pS, McKinney, L. C. andGallin, E. K. (1988) J. Memb. Biol. 103:41 and Matsuda, H. (1988) J.Physiol. 397:237) but smaller than those of muscarinic K⁺ channels (45pS, Horie, M. and Irisawa, H. (1989) J. Physiol. 408:313) orATP-sensitive channels (80 pS, Horie, M. et al. (1987) J. Physiol.387:251). The relatively long open time of the IRK1 channel, and thetendency of the open time to shorten and of the open probability todecrease with increasing hyperpolarization (FIG. 3a), are alsocharacteristic of the inward rectifiers (Matsuda, H., supra and Ohmori,H. (1980) J. Memb. Biol. 53:143).

IRK1 channels in excised inside-out patches rapidly became inactive(within 0.5-15 min; n=7), which resembles the reported run-down ofinward rectifiers (McCloskey, M. A. and Cahalan, M. D. (1990) J. Gen.Physiol. 95:205, muscarinic K⁺ channels (Ito, H. et al. (1991) J. Gen.Physiol. 95:205 and Kurachi, Y. et al. (1992) Prog. Neurobiol. 39;229)and ATP-sensitive K⁺ channels (Horie, M. et al. (1987) supra andKurachi, Y. et al. (1992), supra). Unlike the muscarinic K⁺ channels(Ito, H. et al. (1991), supra and Kurachi, Y. et al. (1992), supra),however, the IRK1 channel activity is not restored by 100 μM internalGTP-TS. In contrast to ATP-sensitive K⁺ channels, which requiremicromolar concentrations of internal Mg-ATP to prevent run-down, butwhich are blocked by internal ATP at millimolar concentrations (Horie,M. et al. (1987) supra and Kurachi, Y. et al. (1992), supra), the IRK1channel exhibited run-down in solutions containing 2.5 mM or 25 μM ATP.Finally, a specific blocker of ATP-sensitive K⁺ channel, tolbutamide(100 μM, Trube, G. et al. (1986) Pflugers Arch. 407:493) did not affectthe IRK1 current.

The experiments shown in FIG. 3 were carried out as follows. Singlechannel recordings were made in the cell attached or the inside-outexcised patch configuration of the patch clamp, using an EPC7 amplifier(List) at 22°-25° C. The pipette solution was 140 mM KCl, 3 mM MgCl₂, 5mM HEPES (pH 7.4). The bath solution was 110 mM KCl, 10 mM MgCl₂, 2 mMK₂ ATP, 5 mM EDTA, 5 mM HEPES, 25 mM KOH (pH 7.2). Total K⁺concentration was 140 mM, and the free Mg²⁺ concentration was calculatedto be 3 mM. Data were filtered at 1kHz by an 8-pole bessel filter,sampled at 2-8 kHz through an A/D converter TL1, and stored in80386-based computer. Single channel current amplitudes were determinedby fitting lines to recordings at each potential. The slope conductancewas determined from a straight line fitted by eye.

FIG. 3a shows current recordings of single channel activities elicitedby voltage steps to various potentials from 0 mV in the cell attachedmode. The linear leakage and capacitive components were subtracted usingaveraged blank records. At -40 mV, as there were no blank tracesrecorded, records at +40 mV were used as leak templates. The potentialsshown represent the pipette potential, the difference between themembrane potential and the pipette potential is the resting potential ofthe oocyte, which could range between 0 and -10 mV in the high K⁺solution. Scale bars indicate 200 ms and 2.5 pA. Triangles showbeginning and end of voltage steps. FIG. 3b shows a I-V plot of thesingle channel current shown in FIG. 3a. V axis shows the pipettepotential; the straight line was fitted by eye. The slope conductancewas 23 pS.

Thus the properties of the IRK1 channel resemble closely those of inwardrectifier K⁺ channels but differ from ATP-sensitive K⁺ channels ormuscarinic K⁺ channels. A plant K⁺ channel (KAT1 from Arabidopsis) hasbeen found to be inwardly rectifying, but it activates at ahyperpolarized membrane potential (-80 mV) regardless of E_(k)(Schachtman, D. P. et al (1992) Science 258:1654). This plant K⁺channel, thus, differs from IRK1 and classical inward rectifier K⁺channels in the mechanism of inward rectification.

3. Primary Structure of the IRK1 Channel

The nucleotide and deduced amino acid sequence of IRK1 (SEQ. ID NOS: 1and 2) is shown in FIGS. 4a(1)-(3). The IRK1 cDNA was sequenced on bothstrands using Sequenase (USB Corp.). The deduced amino acid sequence(SEQ ID NO. 2) is displayed above the nucleotide sequence (SEQ ID NO. 1)of the coding strand of the IRK1 cDNA. Nucleotide A of the initiationcodon (ATG) is assigned as +1. The IRK1 clone (SEQ ID NO. 1) isapproximately 5.5 kb long and has only one long open reading frame whichpredicts a protein of 428 amino acids (SEQ ID NO. 2). Only part of the3' untranslated sequence is shown. The transmembrane segments M1 and M2are boxed. Also boxed is a segment with sequence similarity to the H5region of the voltage-gated K⁺ channel. There are one potential cAMP andcGMP-dependent protein kinase phosphorylation site (Ser 426), fourpotential protein kinase C phosphorylation sites (Ser 3, Thr 6, Ser 357,Thr 383), and two tyrosine kinase phosphorylation sites (Tyr 242, Tyr366).

Sequence analysis reveals that the structure of IRK1 is similar to thatof an ATP-regulated K⁺ channel, ROMK1 (40% amino acid identity, Ho, K.et al. (1993) Nature 362:31). The amino acid sequences of IRK1 (SEQ IDNO. 2) and ROMK1 (SEQ ID NO. 5) are aligned in FIG. 4b. A vertical lineindicates amino acid identity; a period indicates amino acid similarity.Amino acids within each of the following groups were classified assimilar (one letter code): A, S and T; D and E; N and Q; R and K; I, L,M and V; F, Y and W. Dashes indicate gaps introduced into the sequenceto improve alignment. The proposed transmembrane regions M1 and M2 andthe H5 like segments are boxed. The ATP binding site in ROMK1 (SEQ IDNO. 5) is doubly underlined. By use of Monte Carlo and Needleman-Wunchstatistical analysis, these two sequences were determined to besignificantly related. No other sequences with significant similarity tothe IRK1 amino acid sequence (SEQ ID NO. 2) were detected in the GenBankor EMBL data bases.

FIGS. 4c, 4d and 4e show the alignment of the IRK1 (SEQ ID NO. 2) andother K⁺ channel sequences in the H5 (FIG. 4c), S5 (FIG. 4d) and S6(FIG. 4e) region. Boxed residues are identical to those of the IRK1 atthe corresponding positions. The K⁺ channels included in thesealignments are: voltage-gated K⁺ channels of the Shaker subfamily(Kv1.1/RCK1, Stuhmer, W. et al. (1989) EMBO J. 8:3235), the Shabsubfamily (Kv2.1/drk1, Frech, G. C. et al (1989) Nature 340:642), theShaw subfamily (Kv3.1/NGK2, Yokoyama, S. et al. (1989) FEBS Lett.259:37) and the Shal subfamily (Kv4.1/mouse Shal, Pak, M. D. et al.(1991) Proc. Natl. Acad. Sci. USA 88:4386), a Ca²⁺ -dependent K⁺ channel(slo, Atkinson, N. S. et al. (1991) Science 253:551) and a plant K⁺channel (KAT1, Anderson, J. A. et al. (1992) Proc. Natl. Acad. Sci. USA89:3736).

The nucleotide and deduced amino acid sequences of the IRK1 cDNA clone(SEQ ID NOS. 1 and 2) (FIG. 4a) reveal that the structure of IRK1 issimilar to that of ATP-sensitive K⁺ channel (FIG. 4b), but differentfrom that of voltage-gated K⁺ channels. Hydrophobicity analysisindicates the presence of only two potentially membrane-spanninghydrophobic segments (M1, M2). Although IRK1 has fewer hydrophobicsegments than voltage-gated K⁺ channel polypeptides, extensivesimilarity to the H5 pore region of voltage-gated K⁺ channels (Miller,C. (1991) Science 252:1092) was found for a segment between M1 and M2(FIG. 4c). Several residues of M1 and M2 are also identical tocorresponding residues in S5 and S6 respectively, which are highlyconserved among voltage-gated K⁺ channels (FIGS. 4d and 4e). The IRK1sequence contains no hydrophobic segments that correspond to S1, S2 orS3 of voltage-gated K⁺ channels, and only limited sequence similarity toS4 could be detected in the N-terminal region of IRK1. As no N-terminalsignal sequence was detected, the amino-terminus is proposed to be onthe cytoplasmic side of the membrane.

The membrane topology of the IRK1 channel and the ATP-sensitive K⁺channel is likely to resemble that of the S5, H5 and S6 segments of thevoltage-gated K⁺ channel (see FIG. 6, discussed infra). Thus, these twoK⁺ channels belong to a new family which is related to, but distinctfrom, the superfamily of voltage-gated K⁺ channels.

Distribution of IRK1 mRNA in Mouse Tissues

The expression of IRK1 mRNA in various tissues of the mouse was examinedby Northern blot analysis (FIG. 5, upper panel). A 5.5 Kb mRNA for IRK1was detected in J774 cells, forebrain, cerebellum, heart and skeletalmuscle, but not in kidney. The abundance of this 5.5 Kb mRNA was muchhigher in skeletal muscle, heart and forebrain than in cerebellum.

The Northern blot analysis shown in FIG. 5 was performed as follows.Poly A⁺ RNA was isolated using a Fast Track RNA isolation kit(Invitrogen) from J774 cells or tissues of 10 week old male Balb/c mice,from which the J774 cell line was established. RNA sample concentrationswere determined by absorbance at 260 nm and 3 μg poly A⁺ RNA wasfractionated on 0.7% agarose-formaldehyde gel and transferred to a nylonmembrane. A 3.7 Kb BstX1-Not1 fragment from the 3' end of IRK1 waslabeled with ³² P by random priming (Feinberg, A. B. and Vogelstein, B.(1983) Anal. Biochem. 132:6 and Addendum (1984) ibid., 137:266).Hybridizations were performed as described (Baldwin, T. J. et al.(1991), supra). Filters were washed with 0.1× SSC, 1.0% SDS at 65° C.for 15 min and autoradiographed. A RNA size marker (BRL) was probedseparately with ³² P labeled lambda DNA.

The lanes shown in FIG. 5 represent poly A⁺ RNA from J774 cells (1),mouse forebrain (2), cerebellum (3), heart (4), kidney (5) and legskeletal muscle (6). Forebrain includes cerebral cortex, hippocampus,basal ganglia, thalamus, hypothalamus and olfactory bulb. The positionsof RNA size markers are shown on the right of the blot. A major band of5.5 Kb RNA hybridizing to the IRK1 cDNA is indicated with an arrow.

To control for the integrity of RNA, the same blot was reprobed with aprobe for α-tubulin (FIG. 5, lower panel). The α-tubulin probehybridized to a 1.6 kb RNA.

5. Proposed Membrane Topology of the IRK1 Inward Rectifier K⁺ Channel

By analogy with the proposed membrane topology of voltage-gated K⁺channels, the M1 and M2 segments of the IRK1 channel are proposed to bemembrane-spanning and the H5 sequence in between extends from theextracellular surface into the membrane. A similar membrane topology hasbeen proposed for the ATP-regulated K⁺ channel (Ho, K. et al. (1993)Nature 362:31). Compared with the voltage-gated K⁺ channel, which maycontain an outer shell of S1, S2 and S3 segments in contact with thelipid environment (Tempel, B. et al. (1988) Nature 322:837), the inwardrectifier K⁺ channel represents a more reduced structure correspondingto the inner core of the voltage-gated K⁺ channel.

6. Cloning of a Rat G Protein Coupled Muscarinic Potassium Channel

Because the muscarinic K⁺ channel is expressed in the heart and showsinward rectification properties similar to those of the inward rectifierK⁺ channels encoded by ROMK1 (SEQ. ID NO: 5) and IRK1, (SEQ. ID NO: 2) apublicly available rat heart cDNA library (Roberds, S. L. and Tamkun, M.M. (1991) Proc. Natl. Acad. Sci. USA 88:1798) was screened to identifyclones having homology to these two cDNA clones. Two degenerativeprimers were designed using the published amino acid sequences KDGRCNVQ(IRK1 amino acids 50-57) (SEQ. ID NO: 2) and VFQSIVG (IRK1 amino acids162-168) (SEQ. ID NO: 2). The sequences of the primers are AA(A orG)GAIGGICGITG(C or T)AA(C or T)(C or A)T (SEQ. ID NO: 24) andICCIACIATIGA(T or C)TG(G or A)AAIA (SEQ. ID NO: 25) . PCR and screeningof the cDNA library was carried out as described (Baldwin, T. J. et al.(1991) Neuron 7:471) Briefly, total poly A⁺ RNA was isolated from eitherrat heart or rat brain as described supra. 2 μg poly A⁺ RNA was copiedinto first strand cDNA using oligo(dT) priming and reversetranscriptase. The degenerate primers listed above (SEQ. ID NOS: 24 and25) were used to amplify the first-strand cDNA from either rat heart orbrain. The PCR conditions were 94° C. for 30s, 50° C. for 30s and 72° C.for 90s for 40 cycles; AmpliTaq DNA polymerase (Perkin Elmer Cetus) wasused. The resulting PCR products were run on an agarose gel andfragments approximately 350 bp in size were isolated. These fragmentswere cloned into the Smal site of Bluescript SK⁺ (Stratagene) forsequence analysis. Identical PCR products were isolated from the heartand brain. These cloned PCR fragments were then used to screen the ratheart cDNA library to isolate the corresponding full length cDNAs.

The rat heart cDNA library was screened using standard hybridizationtechniques. The hybridization conditions were 50% formamide, 5× SSC, 1×Denhardt's solution, 20 mM sodium phosphate (pH 7.0), 1% SDS, 100 μg/mlsalmon sperm DNA at 42° C. for 16 hours. The filters were washed in 0.1×SSC, 0.1% SDS at 65° C.

A 4.2 kb clone, named GIRK1, (SEQ. ID NO: 3) was isolated and found toencode a protein that is structurally similar to the proteins encoded byROMK1 (SEQ. ID NO: 5) and IRK1 (SEQ. ID NO: 2).

The nucleotide sequence of GIRK1 was determined by the chain terminationmethod using Sequenase (U.S. Biological Corp.) The nucleotide sequenceof GIRK1 is shown in FIG. 7A. Nucleotide A of the first methionine codon(ATG) is assigned as position +1. Only part of the 3' untranslatedsequence is shown. The GIRK1 clone (SEQ. ID NO: 3) comprisesapproximately 4.2 kilobase pairs. There is a single long open readingframe which predicts a protein of 501 amino acids (SEQ. ID NO: 4).

The deduced amino acid sequence of GIRK1 (SEQ. ID NO: 4) is shown abovethe coding strand of the GIRK1 cDNA (SEQ. ID NO: 3) in FIG. 7a(1)-(3).Although the first methionine in the GIRK1 cDNA sequence (SEQ. ID NO: 3)is at a position corresponding to the first methionine of ROMK1 (SEQ. IDNO: 5) and IRK1, (SEQ. ID NO: 2) no stop codons are present in the samereading frame in the nucleotide sequence (SEQ. ID NO: 3) upstream ofthis methionine codon. This raised the possibility that additional aminoacid sequences might exist at the amino terminus of GIRK1. To addressthis question, five independent cDNA clones from one heart and two braincDNA libraries and more than 20 RACE PCR (rapid amplification of cDNAends polymerase chain reaction, Frohman, M. A. (1990) PCR Protocols,Academic Press, San Diego, pp. 28-38) products generated using twodifferent reverse transcriptases (Invitrogen and Gibco BRL) wereanalyzed. This analysis resulted in an extension of only 19 bases (5'CCTTATTGGTGCTGGTTTG 3') (SEQ. ID NO: 26) at the 5' end of the GIRK1 cDNA(SEQ. ID NO: 3). Nonetheless, when the GIRK1 cDNA (SEQ. ID NO: 3) isinjected into Xenopus oocytes, functional K⁺ channels are produced whichhave electrophysiological properties that closely resemble those of theG protein coupled muscarinic K⁺ channel from the heart (Shown in FIG. 8and discussed infra).

Further support for the assignment of the first methionine shown in FIG.7a as the start of the coding region was obtained by the isolation of ahamster GIRK1 cDNA clone from a cDNA library made with RNA isolated froma publicly available hamster HIT cell line (German, M.S. et al. (1991)Mol. Endocrinol. 5:292). This hamster cDNA contains additional sequencesupstream of the first methionine shown in FIG. 7a (SEQ. ID NO: 3).Multiple stop codons are present about 230 bp upstream of the methionineat position +1 and no other methionine codons appear in this region.Theses data confirm that the entire protein coding sequence is presenton the GIRK1 clone (SEQ. ID NO: 3) shown in FIG. 7a.

The proposed transmembrane segments M1 and M2 are boxed. Also boxed is asegment with sequence similarity to the H5 region of voltage-gated K⁺channels. There are 9 potential protein kinase C phosphorylation sites(Thr64, Ser203, Ser284, Ser357, Ser396, Thr407, Ser424, Thr455 andSer497).

FIG. 7B shows an alignment of the amino acid sequences from GIRK1, (SEQ.ID NO: 4) the G protein coupled muscarinic K⁺ channel, IRK1, (SEQ. IDNO: 2) the inward rectifier K⁺ channel and ROMK1, (SEQ. ID NO: 5) theATP-regulated K⁺ channel. For IRK1 (SEQ. ID NO: 2) and ROMK1, (SEQ. IDNO: 5) only the amino acid sequences which differ from GIRK1 (SEQ. IDNO: 4) are given in single letter code. (:)indicates amino acids thatare identical to those in GIRK1 (SEQ. ID NO: 4). (-) indicates gapsintroduced into the sequence to improve alignment. The proposedtransmembrane regions M1 and M2 and the H5-like segment are boxed. GIRK1(SEQ. ID NO: 4) shares 43% identity with IRK1 (SEQ. ID NO: 37) and 39%identity with ROMK1 (SEQ. ID NO: 5) over the total amino acid sequence.

7. The GIRK1 cDNA Directs the Synthesis of a K⁺ Channel

FIG. 8 shows that the GIRK1 cDNA (SEQ. ID NO: 3) encodes a functional K⁺channel which has electrophysiological properties which closely resemblethose of G protein coupled muscarinic K⁺ channels from the heart.Inwardly rectifying K⁺ currents recorded by two electrode voltage clampfrom Xenopus oocytes injected with GIRK1 cRNA are shown in FIG. 8.

Oocytes were injected with 50 nl of cRNA solution, which containsapproximately 300 ng/ml GIRK1 cRNA and 300 ng/ml m2 muscarinic receptorcRNA (FIG. 8a and 8b), or either, or both 500 ng/ml GIRK1 cRNA and 15ng/ml each of Gα_(i2), β₁ and γ₂ cRNA (FIG. 8c, 8d, 8e, 8f and 8g).Eletrophysiological recordings were carried out 48-96 hours later at22°-25° C. using the two electrode voltage clamp technique (Baldwin. T.J. et al. (1991) Neuron 7:471). Data acquisition and analysis were doneon an 80386-based microcomputer using the p-Clamp program and a TL-1 A/Dconverter (Axon Instruments). Microelectrodes were filled with 3M KCland the resistances were 0.7-1.5M ohm. The bath solution contained 90 mMKCl, 3 mM MgCl₂, 5 mM Hepes (pH7.4) (the 90 mM K⁺ solution). Insolutions with reduced K⁺ concentration, K⁺ was replaced withN-methylglucamine.

As is the case with the muscarinic K⁺ channel in the heart, the GIRK1channel could be activated by muscarinic receptor activation. FIG. 8ashows current traces recorded in 90 mM K⁺ solution from oocytes injectedwith m2 muscarinic receptor and GIRK1 cRNA before and after applicationof 1 μM carbachol. The carbachol-induced currents are the difference ofthe former two sets of traces. The holding potential was 0 mV, andcurrent traces elicited by steps to +50, +20, -10, -40, -70, -100 and 31130 mV are shown. The amplitude of carbachol-induced current at -150 mVranged from 290-760 nA (n=8).

FIG. 8b depicts the current-voltage (I-V) plot of the carbachol-inducedcurrent traces shown in FIG. 8a. Current amplitude at the beginning (∇)and at the end () of the steps were plotted. Following co-injection ofm2 muscarinic receptor cRNA (Bonner, T. I. et al. (1987) Science237:527) and GIRK1 cRNA, bath application of 1 μM carbachol induced aninwardly rectifying current (FIG. 8a and 8b). The time course of theinduced currents, obtained by subtracting the currents before from thoseafter bath application of carbachol, showed slow activation at thebeginning of voltage pulses (FIG. 8a and 8b), a characteristic propertyof muscarinic K⁺ channels (Sakmann, B. et al. (1983) Nature 303:250).This carbachol-induced inwardly rectifying current was not observed wheneither the m2 muscarinic receptor (n=4) or GIRK1 (n=4) cRNA alone wasinjected. As the m2 muscarinic receptor is known to activate G proteins(Gilman, A. G. (1984) Cell 36:577 ), the activation of GIRK1 channel wasmost likely mediated by endogenous oocyte G proteins (Dascal, N. (1987)Crit. Rev. Biochem. 22:317) that were coupled with the m2 receptor.

Because the identity of the endogenous G proteins that coupled m2muscarinic receptors to GIRK1 channels could not be readily determined,the effect of an exogenously introduced G protein trimer composed ofα_(i2) (Jones, D. T. and Reed, R. R. (1987) J. Biol. Chem. 262:14241),β₁ (Fong, H. K. W. et al. (1986) Proc. Natl. Acad. Sci. USA 83:2162) andγ₂ (Gautman, N. et al. (1989) Science 244:971) subunits, on the GIRK1channel was investigated.

Previous studies suggested that muscarinic K⁺ channels are coupled withGα_(i) (Yanti, A. et al. (1987) Science 235:207, Yanti, A. et al. (1988)Nature 336:680, and Brown, A. M. and Birnbaumer, L. (1990) Annu. Rev.Physiol. 52:197) and/or βγ subunits (Logothetis, D. E. et al. (1987)Nature 325:321 and Kurachi, Y. et al. (1992) Progress in Neurobiol.39:229), and that a basal level of muscarinic K⁺ channel activation canbe observed without activating muscarinic receptors in the atrial cell(Ito, H. et al (1991) J. Gen. Physiol. 98:517). When cRNA for m2receptor, Gα_(i2), β₁, γ₂ and GIRK1 were coinjected into oocytes, aninwardly rectifying current, similar to the carbachol-induced current,was observed without activating the m2 muscarinic receptor. Activationof m2 receptor did not significantly increase this current. The largebasal activity and the lack of coupling between the exogenouslyintroduced muscarinic receptors and Gα_(i2), β₁, γ₂ might be explainedby the inappropriate combination or ratio of the injected G proteinsubunits in a heterologous expression system. The inwardly rectifyingcurrent was also observed in oocytes injected with Gα_(i2), β₁, γ₂ andGIRK1 cRNA, but not with either Gα_(i2), β₁, γ₂ or GIRK1 cRNA alone(FIGS. 8c & 8d). These results suggest that GIRK1 channels are activatedby exogenously introduced G protein subunits.

FIG. 8c shows current traces recorded in 90 mM K⁺ solution from oocytesinjected with both GIRK1 and Gα_(i2), β₁, γ₂ cRNA or with either GIRK1or Gα_(i2), β₁, γ₂ cRNA alone. The holding potential and thedepolarization steps are as in FIG. 8a. The amplitude of the GIRK1current at -150 mV after linear-leak subtraction ranged from 600-2700 nA(n=35) .

FIG. 8d depicts the I-V plot of the current traces as shown in FIG. 8c.Current amplitude at the end of the steps were plotted. Symbols are ()both GIRK1 and Gα_(i2), β₁, γ₂ cRNA, (∇) only GIRK1 cRNA and () onlyGα_(i2), β₁, γ₂ cRNA.

As expected for a K⁺ selective inwardly rectifying channel (Sakmann, B.et al. (1983) Nature 303:250), the activation potential of GIRK1 channel(see FIG. 8g) shifted to hyperpolarized potential upon reduction ofextracellular K⁺ concentration (FIGS. 8e and 8f) and the amount of shiftfollows the change in the K⁺ equilibrium potential (E_(K)) as predictedby the Nernst equation (FIG. 8g).

FIG. 8e shows the current traces recorded in 45 mM K⁺ or 20 mM K⁺solution from the same oocytes injected with both GIRK1 and G═_(i2), β₁,γ₂ cRNA as in (FIG. 8c, left panel). The holding potential and thedepolarization steps are as in FIG. 8a.

FIG. 8f depicts the I-V plot of the inwardly rectifying current fromoocytes injected with GIRK1 and G═_(i2), β₁, γ₂ in various external K⁺concentration. Symbols are (): 90 mM K⁺, (∇): 45 mM K⁺, () 20 mM K⁺ and(□) 10 mM K⁺. Current amplitude at the end of the steps were plotted.E_(K) 's in these solutions predicted by the Nernst equation were 0 mV(90 mM K⁺), -17 mV (45 mM K⁺), -37 mV (20 mM K⁺), and -55 mV (10 mM K⁺).(The intracellular K⁺ concentration of oocytes was assumed to be 90 mM).The small outward currents in (FIGS. 8c-f) include some endogenouscurrents of the oocyte.

FIG. 8g depicts a logarithmic plot of extracellular K⁺ concentrationversus activation potential (the potential where the slope ofcurrent-voltage relation starts to increase). Vertical bars showstandard deviation. The mean and standard deviation values are 0±4 (n=9;90 mM), -16±3 (n=5; 45 mM), -33±3 (n=7; 20 mM) and -56±6 (n=2; 10 mM).The straight line shows the equilibrium potential of K⁺ predicted by theNernst equation.

8. The GIRK1 Channel is Activated by GTPγS

To test whether the activation of GIRK1 is caused by direct interactionwith G proteins, the effect of the non-hydrolyzable GTP analogue, GTPγS,applied to the cytoplasmic side of excised patches was examined.

In FIG. 9 oocytes were injected with 50 nl of cRNA, which contains about300 ng/ml GIRK1 channel cRNA, 300 ng/ml m2 muscarinic receptor cRNA, and10 ng/ml each of G═_(i2), β₁, γ₂ cRNA, or 500 ng/ml GIRK1 channel cRNAand 15 ng/ml each of G═_(i2), β₁, γ₂ cRNA. The channel activities wereobserved in the absence of carbachol; similar results have been obtainedfrom oocytes injected with GIRK1 and G═_(i2), β₁, γ₂ cRNA, with orwithout m2 receptor cRNA. Single channel activity was recorded incell-attached or inside-out patches. Currents were recorded with a ListEPC7 amplifier at 22°-25° C. The continuous recordings were stored onVCR, transferred to disc at 2-8 kHz through an A/D converter(Instrutech), digitally filtered at 0.5-2 kHz and analyzed by FETCHANprogram. Patch pipettes had resistances of 1-4M ohm. The pipettesolution (external) was 75 mM K₂ SO₄, 15 mM KCl, 2 mM MgSO₄, 10 μMGdCl₃, 5 mM KOH and 10 mM Hepes (pH 7.4). GdCl₃ was included to suppressstretch channel activity (Yang, X.-C. and Sachs, F. (1989) Science243:1068). GdCl₃ was found to have little effect on the GIRK1 currentrecorded by two electrode voltage clamp. The Mg²⁺ -containing bathsolution (internal) was 72.5 mM K₂ SO₄, 15 mM KCl, 4.4 mM MgSO₄, 2.5 mMK₂ ATP, 5 mM KOH, 5 mM Hepes (pH7.2). Total K⁺ concentration was 170 mM,and free Mg²⁺ concentration was calculated to be 2.5 mM. The Mg²⁺ -freebath solution (internal) was 59 mM K₂ SO₄, 15 mM KCl, 10 mM K₂ EDTA, 12mM KOH, 2.5 mM K₂ ATP, 5 mM Hepes (pH 7.2). For both bath solutions, ahigh concentration of ATP (2.5 mM) was used to suppress ATP sensitivechannels (Ashcroft, S. J. H. and Ashcroft, F. M. (1990) CellularSignalling 2:197). In the cell attached mode, the membrane potential ofthe patch differs from the applied potential (the potential differencebetween the pipette solution and the bath solution, each containing 170mM K⁺) by an amount equivalent to the resting potential of the oocytewhich is close to E_(K). Under this condition the applied potential isapproximately the K⁺ driving force across the patch membrane in both thecell-attached mode and the inside-out excised patch mode, so that thesame channel is expected to exhibit the same single channel currentbefore and after excision of the membrane patch.

In cell-attached recordings from oocytes injected with GIRK1 andG═_(i2), β₁, γ₂ cRNA, single-channel openings were observed in theabsence of activated m2 receptor, as was found for the recordings undertwo electrode voltage-clamp. The single-channel activity decreased afterexcising the membrane patch into a solution containing no GTP, but couldbe restored by applying 100 GTPγS to the cytoplasmic side of themembrane (FIG. 9a).

FIG. 9a shows one minute segments of continuous recordings from amembrane patch initially in cell-attached configuration (left panel),subsequently excised (inside-out) into solution without GTP (middlepanel), and then exposed to 100 μM GTPTS (right panel). Expanded tracesfrom the small segments indicated by triangles are shown below. Theholding potential was -60 mV. Open time histograms were fitted by leastsquares with three exponentials. The mean and standard deviation (n=4)of the three time constants and percentage contributions were (cellattached) 0.26±0.11 ms (21±8%), 1.2±0.3 ms (43±9%) and 7.2±1.0 ms(36±10%); excised plus GTPγS, 0.45±0.35 ms (25+13%), 2.3±0.9 ms (50±11%)and 8.9±4.3 ms (25±13%). The time constant of the muscarinic K⁺ channelwas reported to be 0.96 ms (Ito, H. et al. (1992) J. Gen. Physiol.99:961) -1.4 ms (Sakmann, B. et al. (1993) nature 303:250).

In five patches, the normalized open probability (P_(o)) decreased by 6fold after excising into GTP free solution, and increased by 10 foldafter applying GTPγS (FIG. 9b). FIG. 9b shows the open probabilitymeasured from continuous single channel recordings during cell-attached,excised and GTPγS applied periods. Because the number of channels in thepatch was not known, the open probability was normalized to the P_(o)measured in cell-attached patches. The error bars show standarddeviation. The normalized P_(o) (n=5) was 0.18±0.18 after excising and1.78±0.76 with GTPγS (mean±standard deviation). The decrease uponexcision and the increase following exposure to GTPγS were significantby Students' paired t-test (p<0.05).

The channel activities recorded in the cell-attached configuration andthose recorded in the presence of cytoplasmic GTPγS appeared to comefrom the same channel, because the single channel conductance (42 pS)and the kinetics were similar in these two conditions (see FIG. 9a).These results imply that the activation of GIRK1 is caused by aninteraction with G proteins that are associated with the patch ofmembrane, rather than diffusible second messengers that are activated byG proteins.

9. The GIRK1 Channel is the Muscarinic K⁺ Channel

To see if the expressed GIRK1 channel is similar to the muscarinic K⁺channel in the heart (Sakmann, B. et al., supra and Horie, M. andIrisawa, H. (1991) J. Physiol. 408:313) we measured the single channelconductance and the open time and examined if the inward rectificationof GIRK1 channel depended on internal Mg²⁺, as has been shown for themuscarinic K⁺ channel (Horie, M. and Irisawa, H., supra).

GIRK1 showed a single channel conductance of 42 pS in 170 mM K⁺ (FIGS.9c & 9e), as compared to 41 pS in 150 mM K⁺ for the muscarinic K⁺channel (Horie, M. and Irisawa, H., supra). The open time distributionwas similar to that reported for the muscarinic K⁺ channel. Finally,GIRK1 channels activated by GTPγS showed strong inward rectificationwhich depended on the presence of internal Mg²⁺ ; when Mg²⁺ was removedfrom the bath solution, outward currents were recorded from theinside-out patches (FIGS. 9d and 9e). All of the single channelrecordings were obtained in the presence of 2.5 mM cytoplasmic ATP whichblocks ATP-sensitive K⁺ channels. Taken together, these results showthat the GIRK1 channel corresponds to the muscarinic K⁺ channel.

FIG. 9c shows the recording of single channel activity in the presenceof cytoplasmic Mg²⁺. The single channel conductance was 42±1 pS (n=5).Free Mg²⁺ concentration in the cytoplasmic solution was calculated to be2.5 mM.

FIG. 9d shows recordings in Mg²⁺ -free cytoplasmic solution. Onlyoutward channel events are shown. The inward single channel conductancewas 43±2 pS (n=2) in Mg²⁺ -free solution.

FIG. 9e depicts I-V plots of single channel current from the experimentshown in FIG. 9c: () and FIG. 9d: (◯). Straight lines show the best fithaving a slope conductance of 42 pS for Mg²⁺ and 43 pS for Mg²⁺ -freesolution. The deviation of the outward current amplitude in Mg²⁺ -freesolution from ohmic conductance (dashed line) could be due to residualMg²⁺ which remains near the excised patch of membrane even afterperfusing the bath with Mg²⁺ -free solution.

10. Distribution of GIRK1 mRNA in Various Tissues.

The expression of GIRK1 mRNA was examined by Northern blot analysis.Poly(A)⁺ RNA was isolated from tissues of 12 week old rats and 6 weekold guinea pigs using the Fast Track RNA isolation kit (Invitrogen). Theconcentration of the poly(A)⁺ RNA samples were determined by absorbanceat 260 nm. 3 μg poly(A)⁺ RNA was fractionated on a 0.7%agarose-formaldehyde gel and transferred to a nylon membrane.

In FIG. 10 the lanes represent poly A⁺ RNA from rat forebrain,cerebellum, heart ventricle, atrium, skeletal muscle and liver (FIG.10a), and from guinea pig heart ventricle and atrium (FIG. 10b).Forebrain includes cerebral cortex, hippocampus, basal ganglia,thalamus, hypothalamus and olfactory bulb. The atrium preparationincludes part of the vessel stems.

The blots were probed with ³² P-labelled GIRK1 cDNA (FIG. 10a) or a 363bp PCR fragment, obtained from cDNAs reverse-transcribed from guinea-pigheart RNA, which encodes identical amino acids as those of residues 49through 169 of GIRK1 (FIG. 10b). The probes were labeled using therandom priming method (Fienberg and Vogelstein (1983) Annal. Biochem.132:6 and addendum Annal. Biochem 137:266). Hybridizations wereperformed as described in FIG. 5.

Two major RNA species of approximately 4.5 and 6.0 Kb hybridized to theprobes as indicated by the arrows. The positions of RNA size markers(9.5, 7.5, 4.4, 2.4, 1.4 and 0.2 Kb) are shown on the right of each blotas bars.

The integrity of the RNA samples was verified by ethidium bromidestaining of the gel and by reprobing the same blot with a labeled cDNAfor α-tubulin (1.6 Kb message, data not shown). These controls, however,do not rule out the possibility that the smear seen in the rat atriumlane (FIG. 10a) might be due to partial degradation of RNA; much lesssmearing was evident in the guinea-pig atrium lane (FIG. 10b).

FIG. 10 shows that GIRK1 mRNA (approximately 4.5 Kb and 6.0 Kb) is moreabundant in the heart atrium than in the ventricle (FIG. 10a depicts RNAisolated from rat tissues and FIG. 10b depicts RNA isolated from guineapig tissues). This finding is consistent with the distribution of themuscarinic K⁺ channels determined by electrophysiological studies(Sakmann, B. et al., supra, Breitwieser, G. E. and Szabo, G. (1985)Nature 317:538 and Yatani, A. et al., Science, supra).

GIRK1 mRNA was also detected in forebrain and cerebellum, but not inliver or skeletal muscle in rats (FIG. 10a). G protein coupled K⁺channels have been found in the brain (Vandongen, A. M. J. et al. (1988)Science 242:1433 and Brown, D. A. (1990) Annu. Rev. Physiol. 52:215) andthey are activated by various neurotransmitters, such as substance P(Stanfield, P. R. et al. (1985) Nature 315:498), GABA (Gahwiler, B. H.and Brown, D. A. (1985) Proc. Natl. Acad. Sci. USA 82:1558 and Williams,J. T. et al. (1988) J. Neurosci. 8:3499), somatostatin (Mihara, S. etal. (1987) J. Physiol. 390:335 and Inoue, M. et al. (1987) J. Physiol.407:177), opioid (North, R. A. et al. (1987) Proc. Natl. Acad. Sci. USA84:5487 and Wimpey, T. L. and Chavkin, C. (1991) Neuron 6:281) andacetylcholine (Gerber, U. et al. (1991) J. Neurosci. 11:3861). Thus,GIRK1 channel may play a role in regulating the neuronal activity in thebrain as well as the excitability of the heart.

The above examples show that the GIRK1 cDNA, a new member of theinwardly rectifying K⁺ channel superfamily, encodes the muscarinic K⁺channel in the heart. Like the muscarinic K⁺ channel, the GIRK1 channelis abundant in the atrium and is likely to be activated directly by Gproteins. Moreover, the single-channel conductance, kinetics and inwardrectification properties are all similar to those of the muscarinic K⁺channel of the heart.

11. IRK1 and GIRK1 Channels are Expressed in Insulin Secreting Cells

To investigate whether the IRK1 or GIRK1 K⁺ channel, or both, wereexpressed in insulin secreting cells, a cDNA library made with RNAisolated from the HIT-T15 cell line (ATCC CRL 1777) was screened usingthe IRK1 and GIRK1 sequences as probes. The HIT-T15 cell line is aSyrian hamster insulinoma cell line which was established from a primaryculture of pancreatic islet cells which were transformed with SV40 virus(Santerre, B. F. et al. (1981) Proc. Natl. Acad. Sci. USA 78:4339. Thiscell line is a β cell line and secretes insulin in response to glucoseand glucagon stimulation.

A publicly available HIT-T15 cDNA library (German, M. S. et al. (1991)Mol. Endocrinol. 5:292) was screened as follows. Hybridizations werecarried out under high stringency conditions as follows. Briefly, theIRK1 (SEQ. ID NO: 1) and GIRK1 (SEQ. ID NO: 3) cDNAs were labeled with³² P using the random priming method and hybridized to filterscontaining immobilized colonies. Hybridization conditions were asdescribed in Example 6, supra.

cDNA clones corresponding to both GIRK1 (SEQ. ID NO: 3) and IRK1 (SEQ.ID NO: 1) were isolated from the HIT-T15 library. The identity of thecDNA contained in the colony which hybridized to the rat GIRK1 cDNAprobe was confirmed by DNA sequencing and was found to be a bona fidehamster GIRK1 cDNA.

The finding that both the IRK1 and GIRK1 channels are expressed in aninsulin secreting cell supports the assertion that both of these inwardrectifier K⁺ channels play a role in insulin release from the β cells ofthe pancreas. Further bolstering this hypothesis is the fact thatadrenaline is known to regulate insulin release (Rorsman, P. et al.(1991) Nature 349:77); this suggests that GIRK1, the G protein coupledK⁺ channel is involved in insulin release.

While most attention has been focused on the role of the ATP-sensitiveK⁺ channel in insulin secretion, the above results suggest that theATP-sensitive K⁺ channel is not the only inward rectifier K⁺ channelinvolved in insulin secretion.

The finding that the mouse IRK1 (SEQ. ID NO: 1) cDNA and the rat GIRK1(SEQ. ID NO: 3) cDNA can be used to isolate corresponding hamsterhomologues supports the notion that this class of inward rectifier K⁺channels is conserved among the mammals. The present invention'sisolation of first the mouse IRK1 gene and then the rat GIRK1 gene madepossible a finding of conservation among this class of mammalian inwardrectifier K⁺ channels.

12. Materials Testing

After having identified the inward rectifier potassium channelexpression products, IRK1 and GIRK1, extrinsic materials may be assayed.The effect of materials on these K⁺ channel products may preferably bemonitored electrophysiologically, for example, by monitoring changes ofinactivation properties, kinetics, inward rectification properties,alteration in single channel conductance, etc. The same procedures maybe used to screen a battery of materials on the same expression productor on other cells containing either the IRK1 or GIRK1 channels. Theselectivity of a material for a given channel may be determined bytesting the effect of the material on other K⁺ channel types. Forexample, a material can be tested using an expression system whichexpresses the IRK1 channel and the results compared to that seen in anexpression system in which the GIRK1 channel is expressed. In addition,a number of other types of K⁺ channels have been cloned includingvoltage-gated K⁺ channels and ATP-regulated K⁺ channels. Expressionsystems expressing these other K⁺ channels may be used for comparison toselect materials which have a selective effect on either, or both, theIRK1 and GIRK1 channels. Drugs that modulate the IRK1 or GIRK1 channelsselectively are identified as candidates.

A particularly useful method of screening for materials capable ofmodulating the activity of the inward rectifier K⁺ channel expressionproducts, IRK1 and GIRK1 is enabled by the following observation.Oocytes injected with either the IRK1 cRNA or the GIRK1 and G proteincRNAs died when they were exposed to external concentrations of K⁺ of1-10 mM. Cell death or loss of cell viability was determined bymeasuring the membrane resistance of the injected oocyte. A membraneresistance below 0.2 mohms indicates loss of viability. The membraneresistance of a viable oocyte is approximately 1 mohm. Alternatively,loss of viability was determined visually as Xenopus oocytes lose theircharacteristic pigmentation in the animal pole upon cell death.Materials which block the activity of either the IRK1 or GIRK1 K⁺channels prevent cell death thereby enabling a rapid and easy method toscreen materials for the ability to modulate these K⁺ channels.

This method is practiced by injecting oocytes with either the IRK1 cRNAor the GIRK1 and G protein cRNAs as described supra with the exceptionthat the external bath in which the injected oocytes are placed contains1-10 mM K⁺ rather than 50-150 mM K⁺. As discussed supra, isotonic K⁺ foroocytes is approximately 90 mM K⁺. Injected oocytes are typicallysuspended in solutions containing 50-150 mM K⁺, most preferably 90-100mM K⁺ except when one is practicing the above described screeningmethod. A suitable control is always run in parallel with oocytesinjected with either the IRK1 cRNA or the GIRK1 and G protein cRNAs tocontrol for variations in viability between different batches ofuninjected oocytes. Suitable controls include oocytes injected withwater or with cRNA encoding another K⁺ channel such as a voltage-gatedK⁺ channel.

Additionally, the above screening method may also be practiced usingvertebrate or invertebrate cells transfected with DNA encoding the IRK1gene. The transfected cells which express the IRK1 channel are exposedto culture medium containing 1-10 mM K⁺. Materials are screened for theability to prevent cell death. Cell death is detected by the failure ofthe cultured cells to divide.

13. Drug Testing in Disease

Materials comprising drugs identified by the assays described supra asbeing candidates selective for either the IRK1 or GIRK1 K⁺ channels maybe tested in vivo for efficacy in appropriate animal models, forexample, for their ability to regulate the heart beat in that animal orto control the release of insulin. The route of administration of thedrugs can be oral, parental, or via the rectum, and the drug could beadministered alone as principals, or in combination with other drugs,and at regular intervals or as a single bolus, or as a continuousinfusion in standard pharmaceutical formulations. Drugs described supraare also tested in in vitro assays for their ability to modulate theactivity of physiologic functions mediated by the IRK1 or GIRK1 K⁺channels.

14. A Treatment Protocol

Materials identified as a candidate by the assays described above aretested for safety in humans as per Federal guidelines. These candidatesdescribed supra are administered via standard pharmaceuticalformulations to patients with diseases, again either orally,parenterally, rectally, alone or in combination, at regular intervals oras a single bolus, or as a continuous infusion, for modulating IRK1 orGIRK1 K⁺ channels in cells, thus impacting physiological functionsregulated by the activity of these channels.

Notwithstanding that reference has been made to particular preferredembodiments, it will be further understood that the present invention isnot to be construed as limited to such, rather to the lawful scope ofthe appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 26                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2311 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 338..1624                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 596..661                                                        (D) OTHER INFORMATION: /note="Region encoding M1                              segment."                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 731..781                                                        (D) OTHER INFORMATION: /note="Region encoding H5                              segment."                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 806..871                                                        (D) OTHER INFORMATION: /note="Region encoding M2                              segment."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTGCTTCGGCTCATTCTCTTTCACAAAAACCACTGGATCTTACATGCTTCTGTAATCC CC60               ACTTCCACTCCATGTCCCCATGATCCTGTACCAGCAACAGGACAAGTTCTCTGGATGTCA120               GCTGAGTTACTAAGGTAACTTTGCTGGTCAAAAGAACCCCAAGGTTCTCGGAAGCATCCA180               TCTCTCCTCATTAATAAATATATATATTAATT ATATATATATATATAATTTTTTTTGGTG240              TGTCTTCACCGAACATTCAAAACTGTTTCTTCTAAGGGTTTTGCAAAAACTCAGACTGTT300               TTCTAAAGCAGAAACACTGGCGTCCCCAGCGGAAGCAATGGGCAGTGTGAGAACC355                     MetGlySerValArgThr                                                           15                                                                            AACCGCTACAGCATCGTCTCTTCGGAGGAAGATGGCATGAAGCTGGCC403                           As nArgTyrSerIleValSerSerGluGluAspGlyMetLysLeuAla                             101520                                                                        ACTATGGCAGTTGCCAATGGCTTTGGGAATGGCAAGAGTAAAGTCCAT451                           Th rMetAlaValAlaAsnGlyPheGlyAsnGlyLysSerLysValHis                             253035                                                                        ACCCGACAACAGTGCAGGAGCCGCTTTGTGAAGAAAGATGGTCATTGC499                           ThrAr gGlnGlnCysArgSerArgPheValLysLysAspGlyHisCys                             404550                                                                        AATGTTCAGTTTATCAACGTGGGTGAGAAGGGACAGAGGTACCTGGCA547                           AsnValGlnPh eIleAsnValGlyGluLysGlyGlnArgTyrLeuAla                             55606570                                                                      GACATCTTTACTACCTGTGTCGACATCCGCTGGAGGTGGATGCTGGTT595                           AspIl ePheThrThrCysValAspIleArgTrpArgTrpMetLeuVal                             758085                                                                        ATCTTCTGTCTTGCCTTCGTGCTCTCCTGGCTGTTCTTTGGCTGTGTG643                           Il ePheCysLeuAlaPheValLeuSerTrpLeuPhePheGlyCysVal                             9095100                                                                       TTTTGGTTGATAGCCCTGCTCCATGGGGATCTAGATACTTCTAAAGTG691                           Ph eTrpLeuIleAlaLeuLeuHisGlyAspLeuAspThrSerLysVal                             105110115                                                                     AGCAAAGCATGCGTGTCGGAGGTCAACAGCTTCACGGCTGCCTTCCTC739                           SerLy sAlaCysValSerGluValAsnSerPheThrAlaAlaPheLeu                             120125130                                                                     TTCTCCATCGAGACCCAGACAACCATTGGCTATGGTTTCAGGTGTGTG787                           PheSerIleGl uThrGlnThrThrIleGlyTyrGlyPheArgCysVal                             135140145150                                                                  ACAGACGAGTGCCCAATTGCTGTCTTCATGGTGGTATTCCAGTCAATT835                           ThrAs pGluCysProIleAlaValPheMetValValPheGlnSerIle                             155160165                                                                     GTAGGCTGCATCATTGACGCCTTCATCATTGGTGCAGTCATGGCGAAG883                           Va lGlyCysIleIleAspAlaPheIleIleGlyAlaValMetAlaLys                             170175180                                                                     ATGGCAAAGCCAAAGAAGAGAAATGAGACTCTTGTCTTCAGTCACAAT931                           Me tAlaLysProLysLysArgAsnGluThrLeuValPheSerHisAsn                             185190195                                                                     GCTGTGATTGCCATGAGGGATGGCAAACTCTGCTTGATGTGGAGAGTG979                           AlaVa lIleAlaMetArgAspGlyLysLeuCysLeuMetTrpArgVal                             200205210                                                                     GGTAACCTTCGAAAGAGCCACCTTGTGGAAGCTCATGTCCGGGCACAG1027                          GlyAsnLeuAr gLysSerHisLeuValGluAlaHisValArgAlaGln                             215220225230                                                                  CTTCTCAAATCTAGGATCACTTCAGAAGGGGAGTATATCCCTTTGGAC1075                          LeuLe uLysSerArgIleThrSerGluGlyGluTyrIleProLeuAsp                             235240245                                                                     CAGATAGACATCAATGTTGGTTTTGATAGTGGAATTGACCGCATATTT1123                          Gl nIleAspIleAsnValGlyPheAspSerGlyIleAspArgIlePhe                             250255260                                                                     CTAGTGTCCCCCATCACTATCGTTCACGAAATAGATGAAGACAGCCCT1171                          Le uValSerProIleThrIleValHisGluIleAspGluAspSerPro                             265270275                                                                     TTATATGACTTGAGTAAGCAGGACATTGACAATGCAGACTTTGAAATT1219                          LeuTy rAspLeuSerLysGlnAspIleAspAsnAlaAspPheGluIle                             280285290                                                                     GTTGTCATACTGGAAGGCATGGTGGAGGCGACTGCCATGACAACTCAA1267                          ValValIleLe uGluGlyMetValGluAlaThrAlaMetThrThrGln                             295300305310                                                                  TGCCGGAGTTCGTATCTGGCCAATGAAATTCTCTGGGGTCACCGCTAT1315                          CysAr gSerSerTyrLeuAlaAsnGluIleLeuTrpGlyHisArgTyr                             315320325                                                                     GAGCCAGTGCTCTTTGAAGAGAAACACTACTATAAAGTAGACTATTCA1363                          Gl uProValLeuPheGluGluLysHisTyrTyrLysValAspTyrSer                             330335340                                                                     AGATTCCATAAGACTTATGAAGTACCTAACACCCCCCTTTGTAGTGCC1411                          Ar gPheHisLysThrTyrGluValProAsnThrProLeuCysSerAla                             345350355                                                                     AGAGACTTAGCAGAGAAGAAATACATCCTTTCAAATGCAAATTCATTT1459                          ArgAs pLeuAlaGluLysLysTyrIleLeuSerAsnAlaAsnSerPhe                             360365370                                                                     TGCTATGAAAATGAAGTTGCCCTAACAAGCAAAGAGGAAGAGGAGGAT1507                          CysTyrGluAs nGluValAlaLeuThrSerLysGluGluGluGluAsp                             375380385390                                                                  AGTGAGAACGGAGTCCCAGAGAGCACAAGCACAGACTCACCTCCTGGC1555                          SerGl uAsnGlyValProGluSerThrSerThrAspSerProProGly                             395400405                                                                     ATAGATCTCCACAACCAGGCAAGCGTACCTCTAGAGCCCAGGCCCTTA1603                          Il eAspLeuHisAsnGlnAlaSerValProLeuGluProArgProLeu                             410415420                                                                     AGGCGAGAATCGGAGATATGACTGGCTGATTCCGTCTTTGGAATACTT1651                          Ar gArgGluSerGluIle                                                           425                                                                           ACTTTGCTACACAGCCTGACGTTGGTCAGAGGTCCGAGACAGTTATACAGACCATGGTAC1711              TGGTCGAGAGGTGGGTGAAAGCAAGCAGCCACAAGAGACTAAGGCTAGCACAAAGGTTTC1771              AAGGAAAGA CTAAGCTGGATGACTGATGTAAAGAGCTTTGCAGGCCTCCAAGAGACATGA1831             TGGCACATATCTGTTGTAGTATAAGTTATGGGGTTTTTAATGTATTGTTTTGTGTTTTTA1891              CAAAACTTGAATATGCAGGCAAGCCTCAGTTTGGGTACATGACTTACCTGGA ATGCTTCT1951             CTTTAGGGGAACAAGAGTGATTTTAATGGCATAACACAGGCAAGACTCTGCCTTAATTTT2011              TTGAAAAGCTGCTAACTACATGAACACGAACTGTATTTTTATTGCAGTGTAGTTTATCTT2071              TTACATAACGTTAAGACGTCAGTGTTG AGCATTGTTGAAAGCGCAACACAGGCAAGACTC2131             TTGCCTTAATTTTTTGAAAAGCTGCTAACTACATGAACACGAACTGTATTTTTATTGCAG2191              TGTAGTTTATTTACATAACGTTAAGACGTCAGTGTTGAGCATTGTTGAAAGCGCACAGTG2251              T GCTTTAAAGCATCAAGTATTTGGCTATTAACTGCCAAAAATGAAACTGATTTTCTGAGG2311             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 428 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyS erValArgThrAsnArgTyrSerIleValSerSerGluGlu                             151015                                                                        AspGlyMetLysLeuAlaThrMetAlaValAlaAsnGlyPheGlyAsn                              2 02530                                                                       GlyLysSerLysValHisThrArgGlnGlnCysArgSerArgPheVal                              354045                                                                        LysLysAspGlyHisCysAsnValGl nPheIleAsnValGlyGluLys                             505560                                                                        GlyGlnArgTyrLeuAlaAspIlePheThrThrCysValAspIleArg                              657075 80                                                                     TrpArgTrpMetLeuValIlePheCysLeuAlaPheValLeuSerTrp                              859095                                                                        LeuPhePheGlyCysValPheTrpLeuIleAlaLeuLeuHis GlyAsp                             100105110                                                                     LeuAspThrSerLysValSerLysAlaCysValSerGluValAsnSer                              115120125                                                                     PheThrA laAlaPheLeuPheSerIleGluThrGlnThrThrIleGly                             130135140                                                                     TyrGlyPheArgCysValThrAspGluCysProIleAlaValPheMet                              145150 155160                                                                 ValValPheGlnSerIleValGlyCysIleIleAspAlaPheIleIle                              165170175                                                                     GlyAlaValMetAlaLysMetAl aLysProLysLysArgAsnGluThr                             180185190                                                                     LeuValPheSerHisAsnAlaValIleAlaMetArgAspGlyLysLeu                              195200 205                                                                    CysLeuMetTrpArgValGlyAsnLeuArgLysSerHisLeuValGlu                              210215220                                                                     AlaHisValArgAlaGlnLeuLeuLysSerArgIleThrSerGluGly                              2 25230235240                                                                 GluTyrIleProLeuAspGlnIleAspIleAsnValGlyPheAspSer                              245250255                                                                     GlyI leAspArgIlePheLeuValSerProIleThrIleValHisGlu                             260265270                                                                     IleAspGluAspSerProLeuTyrAspLeuSerLysGlnAspIleAsp                              275 280285                                                                    AsnAlaAspPheGluIleValValIleLeuGluGlyMetValGluAla                              290295300                                                                     ThrAlaMetThrThrGlnCysArgSerSerTy rLeuAlaAsnGluIle                             305310315320                                                                  LeuTrpGlyHisArgTyrGluProValLeuPheGluGluLysHisTyr                              325330 335                                                                    TyrLysValAspTyrSerArgPheHisLysThrTyrGluValProAsn                              340345350                                                                     ThrProLeuCysSerAlaArgAspLeuAlaGluLysLysTyr IleLeu                             355360365                                                                     SerAsnAlaAsnSerPheCysTyrGluAsnGluValAlaLeuThrSer                              370375380                                                                     LysGluGluGluG luAspSerGluAsnGlyValProGluSerThrSer                             385390395400                                                                  ThrAspSerProProGlyIleAspLeuHisAsnGlnAlaSerValPro                              40 5410415                                                                    LeuGluProArgProLeuArgArgGluSerGluIle                                          420425                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1827 base pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 59..1564                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 314..379                                                        (D) OTHER INFORMATION: /note="Region encoding M1                              segment."                                                                     ( ix) FEATURE:                                                                (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 455..505                                                        (D) OTHER INFORMATION: /note="Region encoding H5                              segment."                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 530..595                                                        (D) OTHER INFORMATION: /note="Region encoding M2                              segment."                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAGCGCTTGGCTCCTGCGCCTCCGCTTCGTGTTTGAATCTGGATCTCCCCTCCGTATT58                  ATGTCTGCACTCCGAAGGAAATTTGGGGACGATTACCAGGTAGTGACC106                           MetSerAlaLeuArgArgLysPheGlyAspAspTyr GlnValValThr                             151015                                                                        ACTTCGTCCAGCGGTTCGGGCTTGCAGCCCCAGGGGCCAGGACAGGGC154                           ThrSerSerSerGlySerGlyLeuGlnProGln GlyProGlyGlnGly                             202530                                                                        CCACAGCAGCAGCTTGTACCCAAGAAGAAACGGCAGCGGTTCGTGGAC202                           ProGlnGlnGlnLeuValProLysLysLysArg GlnArgPheValAsp                             354045                                                                        AAGAACGGTCGGTGCAATGTGCAGCACGGCAACCTGGGCAGCGAGACC250                           LysAsnGlyArgCysAsnValGlnHisGlyAsnLeu GlySerGluThr                             505560                                                                        AGTCGCTACCTTTCCGACCTCTTCACTACCCTGGTGGATCTCAAGTGG298                           SerArgTyrLeuSerAspLeuPheThrThrLeuValAspLeu LysTrp                             65707580                                                                      CGTTGGAACCTCTTTATCTTCATCCTCACCTACACCGTGGCCTGGCTC346                           ArgTrpAsnLeuPheIlePheIleLeuThrTyrThr ValAlaTrpLeu                             859095                                                                        TTCATGGCGTCCATGTGGTGGGTGATCGCTTATACCCGGGGCGACCTG394                           PheMetAlaSerMetTrpTrpValIleAlaTyr ThrArgGlyAspLeu                             100105110                                                                     AACAAAGCCCATGTCGGCAACTACACTCCCTGTGTGGCCAATGTCTAT442                           AsnLysAlaHisValGlyAsnTyrThrProCys ValAlaAsnValTyr                             115120125                                                                     AACTTCCCCTCTGCCTTCCTTTTCTTCATCGAGACCGAGGCCACCATC490                           AsnPheProSerAlaPheLeuPhePheIleGluThr GluAlaThrIle                             130135140                                                                     GGCTATGGCTACCGCTACATCACCGACAAGTGCCCCGAGGGCATCATC538                           GlyTyrGlyTyrArgTyrIleThrAspLysCysProGluGly IleIle                             145150155160                                                                  CTTTTCCTTTTCCAGTCCATCCTTGGCTCCATCGTGGACGCTTTCCTC586                           LeuPheLeuPheGlnSerIleLeuGlySerIleVal AspAlaPheLeu                             165170175                                                                     ATCGGCTGCATGTTCATCAAGATGTCCCAGCCCAAAAAGCGCGCCGAG634                           IleGlyCysMetPheIleLysMetSerGlnPro LysLysArgAlaGlu                             180185190                                                                     ACCCTCATGTTTAGCGAGCATGCGGTTATTTCCATGAGGGACGGAAAA682                           ThrLeuMetPheSerGluHisAlaValIleSer MetArgAspGlyLys                             195200205                                                                     CTCACTCTCATGTTCCGGGTGGGCAACCTGCGCAACAGCCACATGGTC730                           LeuThrLeuMetPheArgValGlyAsnLeuArgAsn SerHisMetVal                             210215220                                                                     TCCGCGCAGATCCGCTGCAAGCTGCTCAAATCTCGGCAGACACCTGAG778                           SerAlaGlnIleArgCysLysLeuLeuLysSerArgGlnThr ProGlu                             225230235240                                                                  GGTGAGTTTCTACCCCTTGACCAACTTGAACTGGATGTAGGTTTTAGT826                           GlyGluPheLeuProLeuAspGlnLeuGluLeuAsp ValGlyPheSer                             245250255                                                                     ACAGGGGCAGATCAACTTTTTCTTGTGTCCCCTCTCACCATTTGCCAC874                           ThrGlyAlaAspGlnLeuPheLeuValSerPro LeuThrIleCysHis                             260265270                                                                     GTGATTGATGCCAAAAGCCCCTTTTATGACCTATCCCAGCGAAGCATG922                           ValIleAspAlaLysSerProPheTyrAspLeu SerGlnArgSerMet                             275280285                                                                     CAAACTGAACAGTTCGAGGTGGTCGTCATCCTGGAAGGCATCGTGGAA970                           GlnThrGluGlnPheGluValValValIleLeuGlu GlyIleValGlu                             290295300                                                                     ACCACAGGGATGACTTGTCAAGCTCGAACATCATACACCGAAGATGAA1018                          ThrThrGlyMetThrCysGlnAlaArgThrSerTyrThrGlu AspGlu                             305310315320                                                                  GTTCTTTGGGGTCATCGTTTTTTCCCTGTAATTTCTTTAGAAGAAGGA1066                          ValLeuTrpGlyHisArgPhePheProValIleSer LeuGluGluGly                             325330335                                                                     TTCTTTAAAGTCGATTACTCCCAGTTCCATGCAACCTTTGAAGTCCCC1114                          PhePheLysValAspTyrSerGlnPheHisAla ThrPheGluValPro                             340345350                                                                     ACCCCTCCGTACAGTGTGAAAGAGCAGGAAGAAATGCTTCTCATGTCT1162                          ThrProProTyrSerValLysGluGlnGluGlu MetLeuLeuMetSer                             355360365                                                                     TCCCCTTTAATAGCACCAGCCATAACCAACAGCAAAGAAAGACACAAT1210                          SerProLeuIleAlaProAlaIleThrAsnSerLys GluArgHisAsn                             370375380                                                                     TCTGTGGAGTGCTTAGATGGACTAGATGACATTAGCACAAAACTTCCA1258                          SerValGluCysLeuAspGlyLeuAspAspIleSerThrLys LeuPro                             385390395400                                                                  TCGAAGCTGCAGAAAATTACGGGGAGAGAAGACTTTCCCAAAAAACTC1306                          SerLysLeuGlnLysIleThrGlyArgGluAspPhe ProLysLysLeu                             405410415                                                                     CTGAGGATGAGTTCTACAACTTCAGAAAAAGCCTATAGTTTGGGTGAT1354                          LeuArgMetSerSerThrThrSerGluLysAla TyrSerLeuGlyAsp                             420425430                                                                     TTGCCCATGAAACTCCAACGAATAAGTTCGGTTCCTGGCAACTCTGAA1402                          LeuProMetLysLeuGlnArgIleSerSerVal ProGlyAsnSerGlu                             435440445                                                                     GAAAAACTGGTATCTAAAACCACCAAGATGTTATCAGATCCCATGAGC1450                          GluLysLeuValSerLysThrThrLysMetLeuSer AspProMetSer                             450455460                                                                     CAGTCTGTGGCCGATTTGCCACCGAAGCTTCAAAAGATGGCTGGAGGA1498                          GlnSerValAlaAspLeuProProLysLeuGlnLysMetAla GlyGly                             465470475480                                                                  CCTACCAGGATGGAAGGGAATCTTCCAGCCAAACTAAGAAAAATGAAC1546                          ProThrArgMetGluGlyAsnLeuProAlaLysLeu ArgLysMetAsn                             485490495                                                                     TCTGACCGCTTCACATAGCAAAACACCCCATTAGGCATTATTTCATGTTTTGATT1601                   SerAspArgPheThr                                                               500                                                                           TA GTTTTAGTCCAATATTTGGCTGATAAGATAATCCTCCCCGGGAAATCTGAGAGGTCTA1661             TCCCAGTCTGGCAAATTCATCAGAGGACTCTTCATTGAAGTGTTGTTACTGTGTTGAACA1721              TGAGTTACAAAGGGAGGACATCATAAGAAAGCTAATAGTTGGCATG TATTATCACATCAA1781             GCATGCAATAATGTGCAAATTTTGCATTTAGTTTTCTGGCATGATT1827                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 501 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                      MetSerAlaLeuArgArgLysPheGlyAspAspTyrGlnValValThr                              151015                                                                        ThrSerSerSerGlySerGlyLeuGlnProGlnGlyProG lyGlnGly                             202530                                                                        ProGlnGlnGlnLeuValProLysLysLysArgGlnArgPheValAsp                              354045                                                                        LysAsn GlyArgCysAsnValGlnHisGlyAsnLeuGlySerGluThr                             505560                                                                        SerArgTyrLeuSerAspLeuPheThrThrLeuValAspLeuLysTrp                              6570 7580                                                                     ArgTrpAsnLeuPheIlePheIleLeuThrTyrThrValAlaTrpLeu                              859095                                                                        PheMetAlaSerMetTrpTrp ValIleAlaTyrThrArgGlyAspLeu                             100105110                                                                     AsnLysAlaHisValGlyAsnTyrThrProCysValAlaAsnValTyr                              115120 125                                                                    AsnPheProSerAlaPheLeuPhePheIleGluThrGluAlaThrIle                              130135140                                                                     GlyTyrGlyTyrArgTyrIleThrAspLysCysProGluGlyIleIle                               145150155160                                                                 LeuPheLeuPheGlnSerIleLeuGlySerIleValAspAlaPheLeu                              165170175                                                                     Ile GlyCysMetPheIleLysMetSerGlnProLysLysArgAlaGlu                             180185190                                                                     ThrLeuMetPheSerGluHisAlaValIleSerMetArgAspGlyLys                              195 200205                                                                    LeuThrLeuMetPheArgValGlyAsnLeuArgAsnSerHisMetVal                              210215220                                                                     SerAlaGlnIleArgCysLysLeuLeuLys SerArgGlnThrProGlu                             225230235240                                                                  GlyGluPheLeuProLeuAspGlnLeuGluLeuAspValGlyPheSer                              245250 255                                                                    ThrGlyAlaAspGlnLeuPheLeuValSerProLeuThrIleCysHis                              260265270                                                                     ValIleAspAlaLysSerProPheTyrAspLeuSerGlnA rgSerMet                             275280285                                                                     GlnThrGluGlnPheGluValValValIleLeuGluGlyIleValGlu                              290295300                                                                     ThrThrGlyMet ThrCysGlnAlaArgThrSerTyrThrGluAspGlu                             305310315320                                                                  ValLeuTrpGlyHisArgPhePheProValIleSerLeuGluGluGly                               325330335                                                                    PhePheLysValAspTyrSerGlnPheHisAlaThrPheGluValPro                              340345350                                                                     ThrProProTyrSerValLys GluGlnGluGluMetLeuLeuMetSer                             355360365                                                                     SerProLeuIleAlaProAlaIleThrAsnSerLysGluArgHisAsn                              370375 380                                                                    SerValGluCysLeuAspGlyLeuAspAspIleSerThrLysLeuPro                              385390395400                                                                  SerLysLeuGlnLysIleThrGlyArgGluAspPheProLysL ysLeu                             405410415                                                                     LeuArgMetSerSerThrThrSerGluLysAlaTyrSerLeuGlyAsp                              420425430                                                                     Leu ProMetLysLeuGlnArgIleSerSerValProGlyAsnSerGlu                             435440445                                                                     GluLysLeuValSerLysThrThrLysMetLeuSerAspProMetSer                              450 455460                                                                    GlnSerValAlaAspLeuProProLysLeuGlnLysMetAlaGlyGly                              465470475480                                                                  ProThrArgMetGluGlyAsnLeu ProAlaLysLeuArgLysMetAsn                             485490495                                                                     SerAspArgPheThr                                                               500                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 391 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Domain                                                          (B) LOCATION: 83..104                                                         (D) OTHER INFORMATION: /note="M1 segment."                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Domain                                                          (B) LOCATION: 131..147                                                        (D) OTHER INFORMATION: /note="H5 segment."                                     (ix) FEATURE:                                                                (A) NAME/KEY: Domain                                                          (B) LOCATION: 156..177                                                        (D) OTHER INFORMATION: /note="M2 segment."                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetGlyAlaSerGluArgSerValPheArgValLeuIleArgAlaLeu                              15 1015                                                                       ThrGluArgMetPheLysHisLeuArgArgTrpPheIleThrHisIle                              202530                                                                        PheGlyArgSerArgGlnArgAlaArg LeuValSerLysGluGlyArg                             354045                                                                        CysAsnIleGluPheGlyAsnValAspAlaGlnSerArgPheIlePhe                              5055 60                                                                       PheValAspIleTrpThrThrValLeuAspLeuLysTrpArgTyrLys                              65707580                                                                      MetThrValPheIleThrAlaPheLeuG lySerTrpPheLeuPheGly                             859095                                                                        LeuLeuTrpTyrValValAlaTyrValHisLysAspLeuProGluPhe                              100 105110                                                                    TyrProProAspAsnArgThrProCysValGluAsnIleAsnGlyMet                              115120125                                                                     ThrSerAlaPheLeuPheSerLeuG luThrGlnValThrIleGlyTyr                             130135140                                                                     GlyPheArgPheValThrGluGlnCysAlaThrAlaIlePheLeuLeu                              145150 155160                                                                 IlePheGlnSerIleLeuGlyValIleIleAsnSerPheMetCysGly                              165170175                                                                     AlaIleLeuAlaLysIle SerArgProLysLysArgAlaLysThrIle                             180185190                                                                     ThrPheSerLysAsnAlaValIleSerLysArgGlyGlyLysLeuCys                              195 200205                                                                    LeuLeuIleArgValAlaAsnLeuArgLysSerLeuLeuIleGlySer                              210215220                                                                     HisIleTyrGlyLysLeuLeuLys ThrThrIleThrProGluGlyGlu                             225230235240                                                                  ThrIleIleLeuAspGlnThrAsnIleAsnPheValValAspAlaGly                              2 45250255                                                                    AsnGluAsnLeuPhePheIleSerProLeuThrIleTyrHisIleIle                              260265270                                                                     AspHisAsnSe rProPhePheHisMetAlaAlaGluThrLeuSerGln                             275280285                                                                     GlnAspPheGluLeuValValPheLeuAspGlyThrValGluSerThr                              290 295300                                                                    SerAlaThrCysGlnValArgThrSerTyrValProGluGluValLeu                              305310315320                                                                  TrpGlyTyrA rgPheValProIleValSerLysThrLysGluGlyLys                             325330335                                                                     TyrArgValAspPheHisAsnPheGlyLysThrValGluValGluThr                               340345350                                                                    ProHisCysAlaMetCysLeuTyrAsnGluLysAspAlaArgAlaArg                              355360365                                                                     MetLys ArgGlyTyrAspAsnProAsnPheValLeuSerGluValAsp                             370375380                                                                     GluThrAspAspThrGlnMet                                                         385390                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaPheTrpTrpAlaValValSerMetThrThrValGlyTyrGlyAsp                              1 51015                                                                       Met                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        SerPheTrpTrpAlaThrIleThrMetThrThrValGlyTyrGlyAsp                             151015                                                                        Ile                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                     (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlyPheTrpTrpAlaValValThrMetThrThrLeuGlyTyrGlyAsp                              1510 15                                                                       Met                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AlaPheTrpTyrThrIleVal ThrMetThrThrLeuGlyTyrGlyAsp                             151015                                                                        Met                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysValTyrPheLeuIleValThrMetSerThrValGlyTyrGlyAsp                              151015                                                                        Val                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AlaLeuTyrTrpSerIleThrThrLeuThrThrThrGlyTyrGly Asp                             151015                                                                        Phe                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      LeuGlyLeuLeuIlePhePheLeuPheIleGlyValIleLeuPheSer                              151015                                                                        SerAlaValTyrPheAla                                                             20                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      LeuGlyLeuLeuIleLeuPheLeuAlaMetGlyIleMe tIlePheSer                             151015                                                                        SerLeuValPhePheAla                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      PheLeuLeuLeuIleIlePheLeuAlaLeuGlyValLeuIlePheAla                              1510 15                                                                       ThrMetIleTyrTyrAla                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                         (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: /note="M1 segment."                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      LeuGlyPheLeuLeuPheSerLeuThrMetAlaIleIleIlePheAla                              1510 15                                                                       ThrValMetPheTyrAla                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       LeuAlaGlnLeuValSerIlePheIleSerValTrpLeuThrAlaAla                             151015                                                                        GlyIleIleHisLeuLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CysThrLysLeuIleSerValThrLeuPheAlaIleHisCysAlaGly                              1 51015                                                                       CysPheAsnTyrLeuIle                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyLysIleValGlySerLeuCysAlaIleAlaGlyValLeuThrIle                              151015                                                                        AlaLeuPr oValProValIleVal                                                     20                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GlyLysIleVa lGlyGlyLeuCysCysIleAlaGlyValLeuValIle                             151015                                                                        AlaLeuProIleProIleIleVal                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlyMetLeuValGlyAlaLeuCysAlaLeuAlaGlyValLeuThrIle                              1 51015                                                                       AlaMetProValProValIleVal                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GlyLysIlePheGlySerIleCysSerLeuSerGlyValLeuValIle                              151015                                                                        AlaLeu ProValProValIleVal                                                     20                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GlyArgThr PheLeuValPhePheLeuLeuValGlyLeuAlaMetPhe                             151015                                                                        AlaSerSerIleProGluIleIle                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AspIlePhePheMetMetPheAsnLeuGlyLeuThrAlaTyrLeuIle                              1 51015                                                                       GlyAsnMetThrAsnLeuValVal                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AARGAGGCGTGYAAYMT17                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..20                                                           (D) OTHER INFORMATION: /note="Nucleotides indicated as                        "W"are inosine."                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      WCCWACWATWGAYTGRAAWA 20                                                       (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CCTTATTGGTGCTGGTTTG 19                                                    

We claim:
 1. An isolated DNA molecule encoding the inward rectifierpotassium channel, the IRK1 gene product (SEQ ID NO: 2).
 2. A celltransfected with the DNA claim
 1. 3. A vector comprising DNA having thesequence of the DNA molecule of claim 1.